Corticotropin releasing factor 2 receptor agonists

ABSTRACT

Isolated corticotropin releasing factor derivatives, and nucleic acids encoding the same, are effective for treating corticotropin releasing factor 2 receptor modulated disorders such as muscular dystrophy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Applications Ser.Nos. 60/349,117, filed Jan. 16, 2002, 60/376,337, filed Apr. 29, 2002,60/388,895 filed Jun. 14, 2002, and 60/411,988 filed Sep. 19, 2002, allof which are herein incorporated by reference in their entirety.

REFERENCE TO A SEQUENCE LISTING

This Application contains a Sequence Listing appendix, provided as apaper copy, as required under 37 CFR §1.821(c), and is hereinincorporated by reference in its entirety, as required by 37 CFR§1.52(e)(5). A copy of the sequence listing is also provided as requiredunder 37 CFR §1.821(e), as a Computer Readable Form (CRF) on a diskette.

FIELD OF INVENTION

This invention relates to the use of novel peptides, and nucleic acidsencoding the same, to treat CRF₂R modulated disorders.

BACKGROUND CRFR and Ligands

There are at least two corticotropin releasing factor (CRF) receptorsidentified to date (CRF₁R and CRF₂R) which belong to G-protein coupledreceptor (GPCR) class. Agonist activation of CRF₁R or CRF₂R leads toG_(αs) activation of adenylate cyclase. Adenylate cyclase catalyzes theformation of cAMP, which in turn has multiple effects including theactivation of protein kinase A, intracellular calcium release andactivation of mitogen-activated protein kinase (MAP kinase). In otherstudies, the enhancement of intracellular inositol triphosphatesynthesis, after agonist activation of CRF receptors, suggests thatCRFRs also couple to G_(αq).

CRF₁R and CRF₂R have been cloned from human, rat, mouse, chicken, cow,catfish, frog and sheep. CRF₁R and CRF₂R each have a unique distributionpatterns. In humans three isoforms, alpha, beta and gamma, of the CRF₂Rreceptor have been cloned. Homologs for alpha and beta CRF₂R have beenidentified in rat.

Several ligands/agonists of the CRFRs are known and includecorticotropin releasing factor (or hormone, CRF, CRH), urocortin I,urocortin II (or stresscopin related peptide), urocortin III (orstresscopin), urotensin I, sauvagine and other related peptides.Corticotropin releasing factor binds to and activates CRF₁R and CRF₂R.CRF is a major modulator of the body's responses to stress. This41-amino acid peptide presides over a panoply of neuronal, endocrine,and immune processes as the primary regulator of thehypothalamus-pituitary-adrenal hormonal axis (HPA axis). In addition,there is substantial sequence homology between all known ligands ofCRFR. Further, two CRF₂R selective ligands have been identified,urocortin II (or stresscopin related peptide) and urocortin III(stresscopin). These peptides have been identified from multiplemammalian and fish species.

The CRF receptors can be distinguished, from non-CRFRs,pharmacologically through the use of receptor selective agonists andantagonists. These selective agonists and antagonist, along with theCRFR knockout mice, have been useful in determining which CRF receptormediates a particular biological response.

The role of CRF₁R has been fairly well established. Mice in which theCRF₁R gene has been ablated (CRF₁R knockout) demonstrate an impairedstress response and reduced anxiety-like behavior. CRF₁R is a majormediator of the HPA axis. Specifically, CRF, which is released from thehypothalamus and transported to the anterior pituitary via thehypothalamic-hypophysial portal system, interacts with the CRF₁R presenton cells located in the anterior pituitary. Agonist activation of theCRF₁R results in release of ACTH from the cells of the anteriorpituitary into the systemic circulation. The released ACTH binds theACTH receptor present on cells located in the adrenal cortex, resultingin the release of adrenal hormones including corticosteroids.Corticosteroids mediate many effects including, but not limited to,immune system suppression via a mechanism, which involves thymic andsplenic atrophy. Thus activation of the CRF₁R indirectly results in thedown-regulation of the immune system via activation of the HPA axis.

The role of CRF₂R is less well established. Mice in which the CRF₂R genehas been ablated (CRF₂R knockout) demonstrate an impaired or reducedfood intake following stimulation with urocortin, lack of vasodilation,but a normal stress response. Experiments with CRF₂R demonstrated thatCRF₂R is responsible for the hypotensive/vasodilatory effects of CRFRagonists and for the reduction in food intake observed followingtreatment of mice with CRFR agonists.

Skeletal Muscle Atrophy and Hypertrophy

In addition, CRF₂R is involved in the modulation of skeletal muscleatrophy and the induction of hypertrophy. Skeletal muscle is a plastictissue, which readily adapts to changes in either physiological demandfor work or metabolic need. Hypertrophy refers to an increase inskeletal muscle mass while skeletal muscle atrophy refers to a decreasein skeletal muscle mass. Acute skeletal muscle atrophy is traceable to avariety of causes including, but not limited to: disuse due to surgery,bed rest, or broken bones; denervation/nerve damage due to spinal cordinjury, autoimmune disease, or infectious disease; glucocorticoid usefor unrelated conditions; sepsis due to infection or other causes;nutrient limitation due to illness or starvation; and space travel.Skeletal muscle atrophy occurs through normal biological processes,however, in certain medical situations this normal biological processresults in a debilitating level of muscle atrophy. For example, acuteskeletal muscle atrophy presents a significant limitation in therehabilitation of patients from immobilizations, including, but notlimited to, those accompanying an orthopedic procedure. In such cases,the rehabilitation period required to reverse the skeletal muscleatrophy is often far longer than the period of time required to repairthe original injury. Such acute disuse atrophy is a particular problemin the elderly, who may already suffer from substantial age-relateddeficits in muscle function and mass, because such atrophy can lead topermanent disability and premature mortality.

Skeletal muscle atrophy can also result from chronic conditions such ascancer cachexia, chronic inflammation, AIDS cachexia, chronicobstructive pulmonary disease (COPD), congestive heart failure, geneticdisorders, e.g., muscular dystrophies, neurodegenerative diseases andsarcopenia (age associated muscle loss). In these chronic conditions,skeletal muscle atrophy can lead to premature loss of mobility, therebyadding to the disease-related morbidity.

Little is known regarding the molecular processes which control atrophyor hypertrophy of skeletal muscle. While the initiating trigger of theskeletal muscle atrophy is different for the various atrophy initiatingevents, several common biochemical changes occur in the affectedskeletal muscle fiber, including a decrease in protein synthesis and anincrease in protein degradation and changes in both contractile andmetabolic enzyme protein isozymes characteristic of a slow (highlyoxidative metabolism/slow contractile protein isoforms) to fast (highlyglycolytic metabolism/fast contractile protein isoforms) fiber switch.Additional changes in skeletal muscle, which occur, include the loss ofvasculature and remodeling of the extracellular matrix. Both fast andslow switch muscle demonstrate atrophy under the appropriate conditions,with the relative muscle loss depending on the specific atrophy stimulior condition. Importantly, all these changes are coordinately regulatedand are switched on or off depending on changes in physiological andmetabolic need.

The processes by which atrophy and hypertrophy occur are conservedacross mammalian species. Multiple studies have demonstrated that thesame basic molecular, cellular, and physiological processes occur duringatrophy in both rodents and humans. Thus, rodent models of skeletalmuscle atrophy have been successfully utilized to understand and predicthuman atrophy responses. For example, atrophy induced by a variety ofmeans in both rodents and humans results in similar changes in muscleanatomy, cross-sectional area, function, fiber type switching,contractile protein expression, and histology. In addition, severalagents have been demonstrated to regulate skeletal muscle atrophy inboth rodents and in humans. These agents include anabolic steroids,growth hormone, insulin like growth factor I, beta-adrenergic agonists,and CRF₂R agonists. Together, these data demonstrate that skeletalmuscle atrophy results from common mechanisms in both rodents andhumans.

While some agents have been shown to regulate skeletal muscle atrophyand are approved for use in humans for this indication, these agentshave undesirable side effects such as hypertrophy of cardiac muscle,neoplasia, hirsutism, androgenization of females, increased morbidityand mortality, liver damage, hypoglycemia, musculoskeletal pain,increased tissue turgor, tachycardia, and edema. Currently, there are nohighly effective and selective treatments for either acute or chronicskeletal muscle atrophy. Thus, there is a continuing need to identifyother therapeutic agents, which treat skeletal muscle atrophy.

Muscular Dystrophies

Muscular dystrophies encompass a group of inherited, progressive muscledisorders, distinguished clinically by the selective distribution ofskeletal muscle weakness. The two most common forms of muscle dystrophyare Duchenne and Becker dystrophies, each resulting from the inheritanceof a mutation in the dystrophin gene, which is located at the Xp21locus. Other dystrophies include, but are not limited to, limb-girdlemuscular dystrophy which results from mutation of multiple genetic lociincluding the p94 calpain, adhalin, γ-sarcoglycan, and β-sarcoglycanloci; fascioscapulohumeral (Landouzyy-Dejerine) muscular dystrophy,myotonic dystrophy, and Emery-Dreifuss muscular dystrophy. The symptomsof Duchenne muscular dystrophy, which occurs almost exclusively inmales, include a waddling gait, toe walking, lordosis, frequent falls,and difficulty in standing up and climbing stairs. Symptoms start atabout 3–7 years of age with most patients confined to a wheelchair by10–12 years and many die at about 20 years of age due to respiratorycomplications. Current treatment for Duchenne muscular dystrophyincludes administration of prednisone (a corticosteroid drug), whichwhile not curative, slows the decline of muscle strength and delaysdisability. Corticosteroids, such as prednisone, are believed to act byblocking the immune cell activation and infiltration which areprecipitated by muscle fiber damage resulting from the disease.Unfortunately, corticosteroid treatment also results in skeletal muscleatrophy which negates some of the potential benefit of blocking theimmune response in these patients. Thus, there is a continuing need toidentify therapeutic agents which slow the muscle fiber damage and delaythe onset of disability in patients with muscular dystrophies, but causea lesser degree of skeletal muscle atrophy than current therapies.

SUMMARY OF THE INVENTION

The present invention provides isolated peptides that are CRF₂Ragonists. Specifically, the invention provides an isolated peptide, ornucleic acid encoding the same, that are CRF, urocortin I, urocortin II,urocortin III, sauvagine, urotensin I or related peptide derivatives.The invention also provides for pharmaceutical composition comprising asafe and effective amount of an isolated peptide of the presentinvention and a pharmaceutically acceptable excipient. The inventionfurther provides a kit comprising an isolated peptide in unit dose formand usage instructions.

The administration of a peptide, or nucleic acid encoding the same,pharmaceutical composition, or kit of the present invention, to asubject in need thereof, is effective for the treatment of CRF₂Rmodulated disorders such as muscle atrophy or wasting. The inventionalso provides for an antibody that is specific to the peptides of thepresent invention. Lastly, the invention provides for the use of apeptide of the present invention, or nucleic acid encoding the same, inthe manufacture of a medicament for the treatment of a CRF₂R modulateddisorder in a subject in need thereof.

The present invention encompasses isolated non-native peptides accordingto the Formula (I):alpha-beta-gamma-delta-epsilon-zeta-eta-theta  (I)wherein:

-   -   (a) alpha comprises a sequence of a formula X₁X₂X₃X₄X₅X₆;        wherein:        -   X₁, X₂ and X₃ are each selected from the group consisting of            nil, A, E, D G, N, P, Q, S, T, and Z;        -   X₄ is selected from the group consisting of F, I, L, P, T,            and V;        -   X₅ is selected from the group consisting of A, I, P, S, T,            and V;        -   X₆ is selected from the group consisting of I, L, M, and N;    -   (b) beta comprises a sequence of a formula SX₈DX₁₀; wherein: X₈        and X₁₀ are each independently selected from the group        consisting of I, L, and V;    -   (c) gamma comprises a sequence of a formula X₁₁X₁₂X₁₃; wherein:        X₁₁ is selected from a group consisting of P, T, V, and S, and        X₁₂ and X₁₃ are each independently selected from the group        consisting of A, Naphthylalanine (Represented as B), C, D, E, F,        G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;    -   (d) delta comprises a sequence of a formula X₁₄X₁₅X₁₆, wherein:        -   X₁₄ is selected from the group consisting of I, L, and M;        -   X₁₅ is selected from the group consisting of L and M; and        -   X₁₆ is selected from the group consisting of S, N, Q, and R;    -   (e) epsilon comprises a sequence of a formula X₁₇X₁₈X₁₉X₂₀X₂₁,        wherein:        -   X₁₇ is selected from the group consisting of V, I, L, T, K,            E, N, and Q;        -   X₁₈ is selected from the group consisting of L, M, V, A, and            T;        -   X₁₉ is selected from the group consisting of I, F, L, and M;        -   X₂₀ is selected from the group consisting of D, E, N, and H;            and        -   X₂₁ is selected from the group consisting of L, V, I, Q, M,            and R;    -   (f) zeta comprises a sequence of a formula X₂₂X₂₃X₂₄X₂₅,        wherein:        -   X₂₂ is selected from the group consisting of nil, A, D, E,            S, and T;        -   X₂₃ is selected from the group consisting of nil, K, and R;        -   X₂₄ is selected from the group consisting of nil, A H, M, N,            Q, T, and Y;        -   X₂₅ is selected from the group consisting of nil, E, D, I,            K, N, Q, and R;    -   (g) eta comprises a sequence of the formula X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁,        wherein:        -   X₂₆ is selected from the group consisting of A, D, G, H, K,            N, Q, and S;        -   X₂₇ is selected from the group consisting of A, E, I, L, M,            and Q;        -   X₂₈ is selected from the group consisting of A, H, K, Q, R,            and V;        -   X₂₉ is selected from the group consisting of A, E, K, N, M,            and Q;        -   X₃₀ is selected from the group consisting of H, K, N, Q, and            R;        -   X₃₁ is selected from the group consisting of A and K;    -   (h) theta comprises a sequence of the formula        X₃₂X₃₃NX₃₅X₃₆X₃₇X₃₈X₃₉X₄₀X₄₁, wherein:        -   X₃₂ is selected from the group consisting of A, E, H, and T;        -   X₃₃ is selected from the group consisting of A, D, E, I, L,            N, Q, R, S, and T;        -   X₃₅ is selected from the group consisting of A and R;        -   X₃₆ is selected from the group consisting of E, H, I, K, L,            N, Q, and R;        -   X₃₇ is selected from the group consisting of F, I, L, M, and            Y;        -   X₃₈ is selected from the group consisting of L, F, and M;        -   X₃₉ is selected from the group consisting of A, D, E, N, and            Q;        -   X₄₀ is selected from the group consisting of A, D, E, H, I,            K, N, Q, R, S, and T;        -   X₄₁ is selected from the group consisting of A, F, I, and V;            and variants thereof.

All documents cited are, in relevant part, incorporated herein byreference; the citation of any document is not to be construed as anadmission that it is prior art with respect to the present invention.

Sequence Listing Description

Table 1 describes various proteins and protein fragment sequences thatbind to CRF receptors. These selected sequences are included with thecorresponding Genbank or Derwent accession number(s) and the animalspecies from which it is reported, as well as accession numbers forrelated nucleotide sequences that encode identical, or nearly identical,amino acid sequences. These known and novel sequences of the inventionare further presented in the sequence listing.

TABLE 1 DESCRIPTION OF THE INVENTION Genbank (GB), Swiss- Prot (SP) orDerwent (D) amino Accession Related Genbank acid No. for (GB) or DerwentSequence SEQ nucleotide (D) Accession Description ID NO: Speciessequence Nos. urocortin I 2 Homo sapiens Fragment of AC109828 (GB)fragment AF038633 AX015619 (GB) (GB) amino AV708591 (GB) acid residuesAV708591 (GB) 83–122 AAZ35707 (D) AAT73432 (D) urocortin II 4 Homosapiens Fragment of fragment AF320560 (GB) amino acid residues 72–109urocortin III 6 Homo sapiens Fragment of AY026949 (GB) fragment AF361943(GB) amino acid residue 118–157 corticotropin 8 Homo sapiens Fragment ofAC090195 (GB) releasing V00571 AC090196 (GB) hormone (GB) amino BC002599(GB) fragment acid residues AC021240 (GB) 154–194 E00245 (GB)corticotropin 10  Ovis sp. E00212 J00803 (GB) releasing (GB) M22853 (GB)factor fragment sauvagine 11  Phyllomedusa P01144 (SP) sauvagei

Glossary of Terms

The following is a list of definitions for terms used herein.

“Agonist” means any compound, including, but not limited to, antibodies,that activates a receptor. For example, CRFR agonists include, but arenot limited to CRF, urocortin, urocortin II, urocortin III, urotensin I,sauvagine and related analogs.

“Antibody”, in its various grammatical forms, means immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site whichspecifically binds an antigen. As used herein, “isolated antibody,”means an antibody which has been partially or completely separated fromthe proteins and naturally occurring organic molecules with which it isnaturally associated.

“Binding affinity” means the propensity for a ligand to interact with areceptor and is inversely related to the dissociation constant for aspecific CRF ligand-CRFR interaction. The dissociation constant can bemeasured directly via standard saturation, competition, or kineticsbinding techniques or indirectly via pharmacological techniquesinvolving functional assays and endpoints.

“Chimeric antibody” means an antibody that contains structural elementsfrom two or more different antibody molecules, i.e., from differentanimal species. Chimeric antibodies include, but are not limited to,antibodies known as “humanized antibodies” which include, but are notlimited to, chimeric antibodies generated by the technique known ascomplementarity determining region grafting.

“CRF” means corticotropin releasing factor which is the same ascorticotropin releasing hormone (CRH). Exemplary CRF peptides includer/h CRF and ovine CRF (see U.S. Pat. No. 4,415,558), and the like.

“CRF analog” means substances which act as ligands of CRFRs. SuitableCRF analogs can be obtained from a variety of vertebrate species andinclude, but are not limited to, substances such as sauvagine (see,e.g., U.S. Pat. No. 4,605,642), urotensin (see, e.g., U.S. Pat. Nos.4,908,352; and 4,533,654), mouse urocortin II, human urocortin-relatedpeptide (Reyes, T. M. et al., Proc. Nat'l Acad Sci 98:2843–2848 (2001)),urocortin (see, e.g., WO 97/00063), human urocortin II (stresscopinrelated peptide), human urocortin III (stresscopin), pufferfish URP 1,pufferfish URP II, urotensin I, and the CRF analogs described in U.S.Pat. Nos: 4,415,558; 4,489,163; 4,594,329; 4,605,642; 5,109,111;5,235,036; 5,278,146; 5,439,885; 5,493,006; 5663292; 5,824,771;5,844,074; and 5,869,450. Specific CRF analogs include hUcnI (humanurocortin I, AF038633 (GB)); hUroII (human urocortin II or stresscopinrelated peptide)(AF320560); hUroIII (human urocortin III or stresscopin,AF361943); hCRF (human corticotropin releasing factor)(V00571(GB)); oCRF(sheep corticotropin releasing factor E00212 (GB)); Svg (sauvagine,P01144 (SP)).

“CRFR agonist” means a compound or molecule which has the ability toactivate CRF₁R, CRF₂R, or both.

“CRFR” means CRF₁R or CRF₂R. The term “CRFR ” also includes truncatedand/or mutated proteins wherein regions of the receptor molecule notrequired for ligand binding or signaling have been deleted or modified.

“CRF₁R” means any isoforms of CRF₁R from any animal species. The CRF₁Rhas previously been referred to as CRF-RA, PC-CRF, CRF, (Perrin, M. H.,et al. Endocrinology 133:3058–3061 (1993), Chen, R., et al. Proc. Natl.Acad. Sci. USA 90:8967–8971 (1993), Chang, C-P. et al., Neuron11:1187–1195 (1993), Kishimoto, T., et al., Proc. Natl. Acad. Sci. USA,92:1108–1112 (1995) and, Vita, N. et al., FEBS Lett. 335: 1–5 (1993)) orthe CRH receptor.

The definition of CRF₁R includes, but is not limited to, those receptorsfor which the cDNA or genomic sequence encoding the receptor has beendeposited in a sequence database. These sequences include AccessionNos.: X72304, E11431, L23332, I92584, T37068, T28968, Q81952, L23333,NM₁₃004382, AF180301, T28970, L25438, L24096, 192586, Q81954, AH006791,NM₁₃007762, X72305, AF054582, Y14036, AF229359, AF229361, AB055434 andL41563. The nucleotide and protein sequences of these receptors areavailable from GenBank or Derwent.

“CRF₂R” means any isoform of CRF₂R from any animal species. CRF₂R hasalso been referred to as HM-CRF, CRF-RB , (Kishimoto, T., et al., Proc.Natl. Acad. Sci. USA, 92:1108–1112 (1995) and Perrin, M. et al. Proc.Natl. Acad. Sci. USA 92:2969–2973 (1995)).

The definition of CRF₂R receptor includes, but is not limited to, thosereceptors for which the DNA sequence encoding the receptor has beendeposited in a sequence database. These sequences include AccessionNos.: U34587, E12752, NM₁₃001883, T12247, T66508, AF011406, AF019381,U16253, T12244, T28972, U17858, NM₁₃009953, Y14037 and AF229360. Thenucleotide and protein sequences of these receptors are available fromGenBank or Derwent.

“Inhibit” means to partially or completely block a particular process oractivity. For example, a compound inhibits skeletal muscle atrophy if iteither completely or partially prevents muscle atrophy.

“Isolated peptide” means a peptide molecule is said to be “isolated”when physical, mechanical or chemical methods are employed to remove thepeptide from cellular constituents that are normally associated with theprotein. A skilled artisan can readily employ standard purificationmethods to obtain an isolated peptide.

“Isolated nucleic acid” means a nucleic acid molecule is substantiallyseparated from contaminant nucleic acid molecules encoding otherpolypeptides. Purification and sequence identification techniques arewell known in the art.

As used herein, two DNA sequences are said to be “operably associated”if the nature of the linkage between the two DNA sequences does not (1)result in the introduction of a frame-shift mutation, (2) interfere withthe ability of a promoter region to direct the transcription of thecoding sequences, or (3) interfere with the ability of the correspondingRNA transcript to be translated into a protein. For example, a codingsequence and regulatory sequences are operably associated when they arecovalently linked in such a way as to place the transcription of thecoding sequence under the influence or control of the regulatorysequences. Thus, a promoter region is operably associated with a codingsequence when the promoter region is capable of effecting transcriptionof that DNA sequence such that the resulting transcript is capable ofbeing translated into the desired peptide.

“Selective agonist” means that the agonist generally has greater,preferably has significantly greater, activity toward a certainreceptor(s) compared with other receptors, not that it is completelyinactive with regard to other receptors.

“Sequence Identity” or “Homology” at the amino acid or nucleotidesequence level is determined by BLAST (Basic Local Alignment SearchTool) analysis using the algorithm employed by the programs blastp,blastn, blastx, tblastn and tblastx (Altschul et al. (1997) NucleicAcids Res. 25, 3389–3402 and Karlin et al. (1990) Proc. Natl. Acad. Sci.USA 87, 2264–2268) which are tailored for sequence similarity searching.The approach used by the BLAST program is to first consider similarsegments, with gaps (non-contiguous) and without gaps (contiguous),between a query sequence and a database sequence, then to evaluate thestatistical significance of all matches that are identified and finallyto summarize only those matches which satisfy a preselected threshold ofsignificance. For a discussion of basic issues in similarity searchingof sequence databases, see Altschul et al. (1994) Nature Genetics 6,119–129. The search parameters for histogram, descriptions, alignments,expect (i.e., the statistical significance threshold for reportingmatches against database sequences), cutoff, matrix and filter (lowcomplexity) are at the default settings. The default scoring matrix usedby blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoffet al. (1992) Proc. Natl. Acad. Sci. USA 89, 10915–10919), recommendedfor query sequences over 85 nucleotides or amino acids in length.

For blastn, the scoring matrix is set by the ratios of M (i.e., thereward score for a pair of matching residues) to N (i.e., the penaltyscore for mismatching residues), wherein the default values for M and Nare +5 and −4, respectively. Four blastn parameters were adjusted asfollows: Q=10 (gap creation penalty); R=10 (gap extension penalty);wink=1 (generates word hits at every wink^(th) position along thequery); and gapw=16 (sets the window width within which gappedalignments are generated). The equivalent Blastp parameter settings wereQ=9; R=2; wink=1; and gapw=32. A Bestfit comparison between sequences,available in the GCG package version 10.0, uses DNA parameters GAP=50(gap creation penalty) and LEN=3 (gap extension penalty) and theequivalent settings in protein comparisons are GAP=8 and LEN=2.

“Skeletal muscle hypertrophy” means an increase in skeletal muscle massor skeletal muscle function or both.

“Skeletal muscle atrophy” means the same as “muscle wasting” and means adecrease in skeletal muscle mass or skeletal muscle function or both.

In describing protein structure and function, reference is made to aminoacids comprising the protein. The amino acids may also be referred to bytheir conventional abbreviations, as shown: A=Ala=Alanine;T=Thr=Threonine; V=Val=Valine; C=Cys=Cysteine; L=Leu=Leucine;Y=Tyr=Tyrosine; I=Ile=Isoleucine; N=Asn=Asparagine; P=Pro=Proline; Q=GlnGlutamine; F=Phe=Phenylalanine; D=Asp=Aspartic Acid; W=Trp=Tryptophan;E=Glu=Glutamic Acid; M=Met=Methionine; K=Lys =Lysine; G=Gly=Glycine;R=Arg=Arginine; S=Ser=Serine; H=His=Histidine. The letterZ=Glx=Pyrrolidone carboxylic acid, is used to indicate N-terminalglutamic acid or glutamine that has formed an internal cyclic lactam.This has been described in the sequence listing under “MODIFIED₁₃RES”feature where appropriate. The letter B is used in the specification todesignate Naphthylalanine, a modification of Alanine in certain peptidesand has been indicated in the sequence listing under the “miscellaneousfeature” in the sequence listing in the peptide sequences where itoccurs. Abbreviation “Ac” has been used to indicate modified acetylatedNH₂-terminus in the specification and has been described under the“MODIFIED₁₃RES” feature where appropriate. The peptides of the inventionare also modified to have amide group at the carboxy-terminus. This isindicated in the sequence listing under “MODIFIED₁₃RES” feature. Inorder to designate a deletion or an absence of an amino acid in contextof the natural homolog, a “-” or “nil” is used throughout theapplication.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe arts of protein chemistry, pharmacology, or molecular biology. Themethods, materials and examples described herein are not intended to belimiting. Other methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention.

Peptides

The present invention encompasses isolated non-native peptides accordingto the Formula (I):alpha-beta-gamma-delta-epsilon-zeta-eta-theta  (I)

In Formula (I), alpha comprises a sequence of a formula X₁X₂X₃X₄X₅X₆;wherein: X₁, X₂ and X₃ are each selected from the group consisting ofnil, A, E, D G, N, P, Q, S, T and Z; X₄ is selected from the groupconsisting of F, I, L, P, T, and V; X₅ is selected from the groupconsisting of A, I, P, S, T and V; and X₆ is selected from the groupconsisting of I, L, M, and N. In one aspect of the invention, alphacomprises a sequence of the formula X₁X₂X₃X₄X₅X₆; wherein X₁ is nil, X₂is selected from the group consisting of D, E and Z; X₃ is selected fromthe group consisting of D, G and N; X₄ is selected from the groupconsisting of L and P; X₅ is selected from the group consisting of P andS; and X₆ is selected from the group consisting of I, L, M and N. In oneembodiment, alpha further comprises the sequence selected from the groupconsisting of -EDLPL (SEQ ID NO: 388), -DNPSL (SEQ ID NO: 389), -DDPPL(SEQ ID NO: 390), -ZGPPI (SEQ ID NO: 391), -PSL, and -IVL, wherein “-”denotes nil. In another embodiment, alpha comprises the sequence -ZGPPI(SEQ ID NO: 391). In another embodiment, alpha comprises the sequence-DNPSL (SEQ ID NO: 389). In another embodiment, alpha comprises thesequence -IVL. In another embodiment, alpha comprises the sequence -PSL.

In another aspect of the invention, alpha comprises the formulaX₁X₂X₃X₄X₅X₆; wherein X₁ is nil; X₂ is nil; X₃ is nil; X₄ is selectedfrom the group consisting of F, I, L, P and V; X₅ is selected from thegroup consisting of A, I, S, T and V; X₆ is L. In one embodiment, alphacomprises a sequence selected from the group consisting of -IVL, -FTL,-LTL, -FAL, VIL, and -PSL. In another embodiment, alpha comprises thesequence -IVL.

In yet another aspect of the invention, alpha comprises a sequenceselected from the group consisting of SQEPPI (SEQ ID NO: 392), SEEPPI(SEQ ID NO: 393), -DNPSL (SEQ ID NO: 389), -IVL, -TKFTL (SEQ ID NO:394), -ZGPPI (SEQ ID NO: 391), SQEIVL (SEQ ID NO: 395), SEEIVL (SEQ IDNO: 396), DNPIVL (SEQ ID NO: 397), TKIVL (SEQ ID NO: 398), ZGIVL (SEQ IDNO: 399), SDNPSL (SEQ ID NO: 401), STKFTL (SEQ ID NO: 402), SZGPPI (SEQID NO: 403), and NDDPPI (SEQ ID NO: 404).

In yet another aspect of the invention, alpha may be preceded by apolyhistidine (HHHHHH SEQ ID NO: 400) or other peptide tag that may beuseful in the purification or detection of the peptides of theinvention.

In Formula (I), beta comprises a sequence of a formula SX₈DX₁₀, whereinX₈ and X₁₀ are each selected from the group consisting of I, L and V. Inone embodiment, beta comprises the sequence selected from the groupconsisting of SIDL (SEQ ID NO: 405), SLDV (SEQ ID NO: 406), SLDL (SEQ IDNO: 407), SIDI (SEQ ID NO: 408), and SIDV (SEQ ID NO: 409). In anotherembodiment beta comprises the sequence further selected from the groupconsisting of SIDL (SEQ ID NO: 405) and SLDV (SEQ ID NO: 406). In yetanother embodiment, beta comprises the sequence SIDL (SEQ ID NO: 405).In yet another embodiment, beta comprises the sequence SLDV (SEQ ID NO:406). In yet another embodiment, beta comprises the sequence SIDV (SEQID NO: 409).

In Formula (I), gamma comprises a sequence of a formula X₁₁X₁₂X₁₃;wherein X₁₁, is P T, V, or S, and X₁₂ and X₁₃ are each selected from thegroup consisting of A, B (Naphthylalanine), C, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, and Y. In one embodiment of the invention X₁₁is P. In another embodiment, gamma comprises the sequence selected fromthe group consisting of PAB, PAF, PAH, PAQ, PAY, PFB, PFE, PFF, PFG,PFH, PFI, PFL, PFQ, PFV, PFW, PFY, PGY, PHB, PHF, PHH, PHQ, PHW, PHY,PIA, PIB, PID, PIE, PIF, PIG, PIH, PII, PIL, PIQ, PIR, PIT, PIV, PIW,PIY, PKY, PLB, PLE, PLF, PLG, PLH, PLI, PLL, PLQ, PLV, PLW, PLY, PNY,PQB, PQF, PQH, PQI, PQL, PQQ, PQV, PQW, PQY, PRY, PSY, PTB, PTE, PTF,PTH, PTI, PTL, PTV, PTW, PTY, PVB, PVY, PWF, PWH, PWQ, PWW, PWY, PYB,PYF, PYH, PYI, PYL, PYQ, PYT, PYV, PYW, PYY, SLE, SLG, SIG, and VIG. Inyet another embodiment of the invention, gamma comprises the sequenceselected from the group consisting of PFE, PFG, PFH, PFQ, PFY, PLE, PLG,PLH, PLQ, PLY, PTE, PTH, PTY, PIE, PIH, PIQ, PIY, PIG, PTN and PTS. Inyet another embodiment, gamma comprises the sequence selected from thegroup consisting of PFE, PFG, PFH, PFQ, PFY, PLE, PLG, PLH, PLQ, PLY,PTE, PTH, PTY, PIE, PIH, PIQ, PIY, PYY, PFE, PTW, PQY, PHY, PII, PIL,PTI, PTF, PTL, PIV, PIT, PTV and PIE. In yet another embodiment, gammacomprises the sequence selected from the group consisting of PIG, PTN,PTS, and PIG. In yet another embodiment, gamma comprises of sequenceselected from PFQ, PYW, PLQ, PIG, PLY, PUY, PTY, PIG, PLL, PLF, and PFF.In yet another embodiment, gamma comprises the sequence PIG. In yetanother embodiment, gamma comprises PFQ.

In Formula (I), delta comprises a sequence of a formula X₁₄X₁₅X₁₆,wherein X₁₄ is selected from the group consisting of I, L, and M; X₁₅ isselected from the group consisting of L and M; and X₁₆ is selected fromthe group consisting of S, N, Q, and R. In one embodiment, deltacomprises a sequence selected from the group consisting of ILS, IMN,LLQ, LLR, and MLR. In one embodiment, delta comprises the sequence LLQor LLR.

In Formula (I), epsilon comprises a sequence of a formulaX₁₇X₁₈X₁₉X₂₀X₂₁, wherein, X₁₇ is selected from the group consisting ofV, I, L, T, K, E, N, and Q; X₁₈ is selected from the group consisting ofL, M, V, A, and T; X₁₉ is selected from the group consisting of I, F, L,and M; X₂₀ is selected from the group consisting of D, E, N, and H; andX₂₁ is selected from the group consisting of L, V, I, Q, M, and R. Inone embodiment, epsilon comprises a sequence selected from the groupconsisting of VLIDL (SEQ ID NO: 410), VLFDV (SEQ ID NO: 411), VLIEI (SEQID NO: 412), ILFNI (SEQ ID NO: 413), LLIEI (SEQ ID NO: 414), LLFNI (SEQID NO: 415), ILLEQ (SEQ ID NO: 416), ILIEI (SEQ ID NO: 417), ILLEI (SEQID NO: 418), TLLEL (SEQ ID NO: 419), KMIEI (SEQ ID NO: 420), KVIEI (SEQID NO: 421), EVLEM (SEQ ID NO: 422), EMIEI (SEQ ID NO: 423), EVIEI (SEQID NO: 424), EAIEI (SEQ ID NO: 425), ETIEI (SEQ ID NO: 426), EIIEI (SEQID NO: 427), ELIEI (SEQ ID NO: 428), NMIEM (SEQ ID NO: 429), NMIHR (SEQID NO: 430), NMIHM (SEQ ID NO: 431), QMMEM (SEQ ID NO: 432), and LLFNI(SEQ ID NO: 433). In one embodiment of the invention, epsilon comprisesthe sequence selected from the group consisting of VLIDL (SEQ ID NO:410), VLFDV SEQ ID NO: 411), ILFNI (SEQ ID NO: 413), LLFNI (SEQ ID NO:415), ILLEQ (SEQ ID NO: 416), TLLEL (SEQ ID NO: 419) and KMIEI (SEQ IDNO: 420). In another embodiment, epsilon comprises the sequence selectedfrom the group consisting of VLIDL (SEQ ID NO: 410), VLFDV (SEQ ID NO:411), ILFNI (SEQ ID NO: 413) and ILLEQ (SEQ ID NO: 416). In yet anotherembodiment, epsilon comprises the sequence selected from the groupconsisting of KMIEI (SEQ ID NO: 420) or ILLEQ (SEQ ID NO: 416). In yetanother embodiment, epsilon comprises the sequence KVIEI (SEQ ID NO:421), KMIEI (SEQ ID NO: 420), ILLEI (SEQ ID NO: 417), ILLEQ (SEQ ID NO:416), or TLLEL (SEQ ID NO: 419). In yet another embodiment, epsiloncomprises the sequence KMIEI (SEQ ID NO: 420). In yet anotherembodiment, epsilon comprises the sequence ILLEQ (SEQ ID NO: 416).

In Formula (I), zeta comprises a sequence of a formula X₂₂X₂₃X₂₄X₂₅,wherein X₂₂ is selected from the group consisting of nil, A, D, E, S andT; X₂₃ is selected from the group consisting of nil, K and R; X₂₄ isselected from the group consisting of nil, A H, M, N, Q, T and Y; andX₂₅ is selected from the group consisting of nil, E, D, I, K, N, Q andR. In one embodiment of the invention, zeta comprises a sequence of theformula X₂₂X₂₃X₂₄X₂₅; wherein X₂₂ is selected from the group consistingof nil, D and E; X₂₃ is selected from the group consisting of nil, K andR; X₂₄ is selected from the group consisting of nil, A H, M, N, Q, T andY; X₂₅ is selected from the group consisting of nil, E, D, I, K, N, Qand R. In another embodiment, zeta comprises a sequence selected fromthe group consisting of SRAE (SEQ ID NO: 434), EKAR (SEQ ID NO: 435),ERAR (SEQ ID NO: 436), EKQE (SEQ ID NO: 437), TKDR (SEQ ID NO: 438),TKAD (SEQ ID NO: 439), AKAR (SEQ ID NO: 440), AKQR (SEQ ID NO: 441),ERQR (SEQ ID NO: 442), AKAE (SEQ ID NO: 443), ERAE (SEQ ID NO: 444),ARQR (SEQ ID NO: 445), EKQR (SEQ ID NO: 446), TKAN (SEQ ID NO: 447),TKAR (SEQ ID NO: 448), EAAR (SEQ ID NO: 449), ERQE (SEQ ID NO: 450),ARAD (SEQ ID NO: 451), EKTQ (SEQ ID NO: 452), ARAR (SEQ ID NO: 453),ARAE (SEQ ID NO: 454), ARQE (SEQ ID NO: 455), AKQE (SEQ ID NO: 456),TRAD (SEQ ID NO: 457), AKAD (SEQ ID NO: 458), TRAR (SEQ ID NO: 459),EKQQ (SEQ ID NO: 520), -RR, -AA, -AAR, -R, -RAR, -A, -AR, -ARA, -R-R,A-AR, A-A-, A-, ARA- and -. In yet another embodiment, zeta comprises asequence selected from the group consisting of EKAR (SEQ ID NO: 435),ERAR (SEQ ID NO: 436), EKQE (SEQ ID NO: 437) and TKDR (SEQ ID NO: 438).In yet another embodiment, zeta comprises EKQE (SEQ ID NO: 437), EKTQ(SEQ ID NO: 452), ARAR (SEQ ID NO: 453), or EKAR (SEQ ID NO: 435).

In Formula (I), eta comprises a sequence of a formulaX₂₆X₂₇X₂₈X₂₉X₃₀X₃₁, wherein: X₂₆ is selected from the group consistingof A, D, G, H, K, N, Q, and S; X₂₇ is selected from the group consistingof A, E, I, L, M and Q; X₂₈ is selected from the group consisting of A,H, K, Q, R and V; X₂₉ is selected from the group consisting of A, E, K,M, N and Q; X₃₀ is selected from the group consisting of H, K, N, Q andR; and X₃₁ is selected from the group consisting of A and K. In oneembodiment of the invention, eta comprises a sequence selected from thegroup consisting of AAREQA (SEQ ID NO: 460); KEKKRK (SEQ ID NO: 461);SQRERA (SEQ ID NO: 462), KEKQQA (SEQ ID NO: 463), and QLAQQA (SEQ ID NO:464) AARNQA (SEQ ID NO: 521), KERNQA (SEQ ID NO: 522), KEKNQA (SEQ IDNO: 523), KQRERA (SEQ ID NO: 524), KERERA (SEQ ID NO: 525) KEKERA (SEQID NO: 526), KEKQRA (SEQ ID NO: 527), AEAAAK (SEQ ID NO: 528), AAHAAA(SEQ ID NO: 529), and HAHAHA (SEQ ID NO: 530). In yet anotherembodiment, eta comprises a sequence selected from the group consistingof AAREQA (SEQ ID NO: 460), and KEKKRK (SEQ ID NO: 461). In yet stillanother embodiment, eta comprises the sequence AAREQA (SEQ ID NO: 460).In yet still another embodiment, eta comprises a sequence selected fromthe group consisting of SQRERA (SEQ ID NO: 462) and KEKQQA (SEQ ID NO:463). In yet another embodiment, eta comprises the sequence KEKQQA (SEQID NO: 463).

In Formula (I), theta comprises a sequence of the formulaX₃₂X₃₃NX₃₅X₃₆X₃₇X₃₈X₃₉X₄₀X₄₁, wherein X₃₂ is selected from the groupconsisting of A, E, H and T; X₃₃ is selected from the group consistingof A, D, E, I, L, N, Q, R, S and T; X₃₅ is selected from the groupconsisting of A and R; X₃₆ is selected from the group consisting of E,H, I, K, L, N, Q and R; X₃₇ is selected from the group consisting of F,I, L, M and Y; X₃₈ is selected from the group consisting of L, F and M;X₃₉ is selected from the group consisting of A, D, E, N and Q; X₄₀ isselected from the group consisting of A, D, E, H, I, K, N, Q, R, S andT; X₄₁ is selected from the group consisting of A, F, I and V. In oneembodiment of the invention, theta comprises a sequence of the formulaX₃₂X₃₃NX₃₅X₃₆X₃₇X₃₈X₃₉X₄₀X₄₁, wherein X₃₂ is selected from the groupconsisting of A, E and T; X₃₃ is selected from the group consisting ofA, D, E, N, Q, S and T; X₃₅ is selected from the group consisting of Aand R; X₃₆ is selected from the group consisting of H, I, L, N, Q and R;X₃₇ is selected from the group consisting of F, I L, M, and Y; X₃₈ isselected from the group consisting of L, F and M; X₃₉ is selected fromthe group consisting of A, D, E, N and Q; X₄₀ is selected from the groupconsisting of nil, A, D, H, Q, R, S and T; X₄₁ is selected from thegroup consisting of I and V. In another embodiment, theta comprises asequence selected from the group consisting of AANRLLLDTV (SEQ ID NO:465), AAQEQILAHV (SEQ ID NO: 466), ANNAELLAEI (SEQ ID NO: 467),ANNAHLLAHI (SEQ ID NO: 468), ANNAKLLAKI (SEQ ID NO: 469), ANNALLLATI(SEQ ID NO: 470), ANNALLLDTI (SEQ ID NO: 471), ANNANLLANI (SEQ ID NO:472), ANNAQLLAHI (SEQ ID NO: 473), ANNAQLLAQI (SEQ ID NO: 474),ANNARILARV (SEQ ID NO: 475), ANNARLLARI (SEQ ID NO: 476), ANNARLLDTI(SEQ ID NO: 477), ANNRLLLATI (SEQ ID NO: 478), ANNRLLLDTI (SEQ ID NO:479), EQNAHIFAHV (SEQ ID NO: 480), EQNAQIFAHV (SEQ ID NO: 481),EQNARIFARV (SEQ ID NO: 482), EQNRIIFDSV (SEQ ID NO: 483), ETNARILARV(SEQ ID NO: 484), HAQAHILAHV (SEQ ID NO: 485), HSNRKIIDIA (SEQ ID NO:486), HSNRKLLDIA (SEQ ED NO: 487), HSNRKLMEII (SEQ ID NO: 488),HTNARILARV (SEQ ID NO: 489), TNNRLLLATV (SEQ ID NO: 490), TNNRLLLDTI(SEQ ID NO: 491), TSNRKLMEII (SEQ ID NO: 492), TTNARILARN (SEQ ID NO:493), TTNARILARV (SEQ ID NO: 494), TTNARLLATV (SEQ ID NO: 495),TTNARLLDRV (SEQ ID NO: 496), TTNARLLDTV (SEQ ID NO: 497), TTNRLLLARV(SEQ ID NO: 498), TTNRLLLATV (SEQ ID NO: 499), TTNRLLLDTV (SEQ ID NO:500), TTQARILARV (SEQ ID NO: 501), and TTVARILARV (SEQ ID NO: 502). Inyet another embodiment, theta comprises a sequence selected from thegroup consisting of TTNARILARV (SEQ ID NO: 494), ANNALLLDTI (SEQ ID NO:471), ANNALLLATI (SEQ ID NO: 470), TTNARLLDTV (SEQ ID NO: 497) andTTNARLLDRV (SEQ ID NO: 496). In yet another embodiment, theta comprisesthe sequence ANNARLLDTI (SEQ ID NO: 477), ANNARLLARI (SEQ ID NO: 476),ANNALLLDTI (SEQ ID NO: 471), ANNALLLATI (SEQ ID NO: 470), TTNARLLDRV(SEQ ID NO: 496), TTNARILARV (SEQ ID NO: 494), ANNRLLLDTI (SEQ ID NO:479), EQNARIFARV (SEQ ID NO: 482), EQNAHIFAHV (SEQ ID NO: 480), andEQNAQIFAHV (SEQ ID NO: 481). One skilled in the art will readilyappreciate that theta encompasses the C-terminus end of the peptide.

In one embodiment, the invention comprises a non-native peptidecomprising the sequence:X₁X₂PSLSIDX₉PX₁₁X₁₂LLRTLLELEKTQSQRERAEQNAX₃₅IFAX₃₉V (SEQ ID NO: 531),wherein: X₁ is selected from nil and D; X₂ is selected from nil and N;X₉ is selected from L and V; X₁₁ is selected from L, and F; X₁₂ isselected from L, F, and Y; X₃₅, is selected from R, H, and Q; X₃₉ isselected from H and R; and variants thereof having at least about 95%sequence identity to said peptide.

The peptides of the invention have also been described as a peptide of41 amino acids with certain preferred sequences. Following peptidestrings have been specifically exemplified: ZGPPISIDLP (SEQ ID NO: 503)for residues X₂–X₁₁, LLRK (SEQ ID NO: 504) for residues X₁₄–X₁₇,IEIEKQEKEKQQA (SEQ ID NO: 505) for residues X₁₉–X₃₁, PSLSID (SEQ ID NO:506) for residues X₄–X₉, and LLRTLLELEKTQSQRERAEQNA (SEQ ID NO: 507) forresidues X₁₄–₃₅.

Variants of the disclosed peptides, and nucleotide sequences encodingthe same, are also encompassed by the present invention. As used herein,“variants,” means those peptides, polypeptides or proteins, ornucleotide sequences encoding the same, that are substantially similarto those peptides described by Formula (I) and which may be used asCRF₂R agonists. A peptide of Formula (I) may be altered in various waysto yield a variant of those encompassed by the present inventionincluding amino acid substitutions, deletions, truncations, insertions,and modifications. Methods for such manipulations are generally known inthe art. For example, variants can be prepared by mutations in thenucleotide sequences encoding the same. Methods for mutagenesis andnucleotide sequence alterations are well known in the art. See, forexample, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488–492; Kunkel etal. (1987) Methods in Enzymol. 154:367–382; U.S. Pat. No. 4,873,192;Walker and Gaastra, eds. (1983) Techniques in Molecular Biology(MacMillan Publishing Company, New York) and the references citedtherein. In one embodiment of the variant, the substitution(s) of thepeptide of Formula (I) is conservative in that it minimally disrupts thebiochemical properties of the variant. Thus, where mutations areintroduced to substitute amino acid residues, positively chargedresidues (H, K, and R) preferably are substituted with positivelycharged residues; negatively charged residues (D and E) preferably aresubstituted with negatively-charged residues; and neutral non-polarresidues (A, F, I, L, M, P, V, and W) preferably are substituted withneutral non-polar residues. In another embodiment of the variant, theoverall charge, structure or hydrophobic/hydrophilic properties of thepeptide can be altered without substantially adversely affecting CRF₂Ragonism. In still another embodiment, the variant is an active fragmentof a peptide of Formula (I). In yet another embodiment of a variant, apeptide of Formula (I) is modified by acetylation, carboxylation,phosphorylation, glycosylation, ubiquitination, and labeling, whetheraccomplished by in vivo or in vitro enzymatic treatment of the proteinor by the synthesis of the peptide using modified amino acids. Commonnon-limiting examples of modifications to amino acids includephosphorylation of tyrosine, serine, and threonine residues; methylationof lysine residue; acetylation of lysine residues; hydroxylation ofproline and lysine residues; carboxylation of glutamic acid residues;glycosylation of serine, threonine, or asparagine residues; andubiquitination of lysine residues. The variant can also include otherdomains, such as epitope tags and His tags (e.g., the peptide can be afusion protein).

In yet another embodiment, peptide mimics of a peptide of Formula (I)are encompassed within the meaning of variant. As used herein, “mimic,”means an amino acid or an amino acid analog that has the same or similarfunction characteristics of an amino acid. Thus, for example, anarginine analog can be a mimic of arginine if the analog contains a sidechain having a positive charge at physiologic pH, as is characteristicof the guanidinium side chain reactive group of arginine. Examples oforganic molecules that can be suitable mimics are listed at Table 1 ofU.S. Pat. No. 5,807,819. Generally, a variant, or nucleic acid sequenceencoding the same, of the present invention will have at least 70%,generally, 80%, preferably up to 90%, more preferably 95%, even morepreferably 97%, still even more preferably 98%, and most preferably 99%sequence identity to its respective native amino acid sequence. Fusionproteins, or N-terminal, C-terminal or internal extensions, deletions,or insertions into the peptide sequence shall not be construed asaffecting homology.

Use of the Peptides of the Invention as CRF₂R Agonists

The peptides of the invention are useful for the treatment of a varietyof diseases, disorders, and conditions that are modulated by CRF₂R or byCRF₂R activity. As used herein, the terms “disease,” disorder” and“condition” are used interchangeably. As used herein, a disorderdescribed by the terms “modulated by CRF₂R,” or “modulated by CRF₂Ractivity” refers to a disorder, condition or disease where CRF₂Ractivity is an effective means of alleviating the disorder or one ormore of the biological manifestations of the disease or disorder; orinterferes with one or points in the biological cascade either leadingto the disorder or responsible for the underlying disorder; oralleviates one or more symptoms of the disorder. Thus, disorders subjectto “modulation” include those for which: (1) The lack of CRF₂R activityis a “cause” of this disorder or one or more of the biologicalmanifestations, whether the activity was altered genetically, byinfection, by irritation, by internal stimulus or by some other cause;(2) The disease or disorder or the observable manifestation ormanifestations of the disease or disorder are alleviated by CRF₂Ractivity (the lack of CRF₂R activity need not be causally related to thedisease or disorder or the observable manifestations thereof); (3) CRF₂Ractivity interferes with part of the biochemical or cellular cascadethat results in or relates to the disease or disorder. In this respect,the CRF₂R activity alters the cascade, and thus controls the disease,condition, or disorder.

In one embodiment of the invention, the peptides of the presentinvention have none or only weak CRF₁R agonist activity. Thus, thepeptides of the present invention are particularly useful for thetreatment of CRF₂R modulated disorders. One such CRF₂R modulateddisorder is skeletal muscle atrophy. Skeletal muscle atrophy may beinduced by disuse due to surgery, bed rest, broken bones;denervation/nerve damage due to spinal cord injury; autoimmune disease;infectious disease; glucocorticoid use for unrelated conditions; sepsisdue to infection or other causes; nutrient limitation due to illness orstarvation; cancer cachexia; chronic inflammation; acquiredimmunodeficiency syndrome (AIDS); cachexia; chronic obstructivepulmonary disease (COPD); congestive heart failure; sarcopenia andgenetic disorders; e.g., muscular dystrophies, neurodegenerativediseases.

In another embodiment, the treatment of a CRF₂R modulated disorderresults in an increase of skeletal mass and function. Diseases andconditions affecting skeletal muscle mass and function include, but notlimited to, skeletal muscle atrophy or wasting including acuteatrophy/wasting resulting from disuse due to illness, surgery, bed restor accident; nerve damage due to spinal cord injury, autoimmune disease,or infectious disease; glucocorticoid use for unrelated conditions;sepsis due to infection or other causes; nutrient limitation due toillness or starvation; and space travel: and chronic atrophy/wastingincluding cancer cachexia, chronic inflammation, AIDS cachexia, COPD,congestive heart failure, genetic disorders, e.g., muscular dystrophies,neurodegenerative diseases and sarcopenia (age associated muscle loss).

In yet another embodiment, the treatment of a CRF₂R modulated disorderincludes disorders affecting bone. Diseases and conditions affectingbone include, but not limited to, bone loss resulting from disuse due toillness, surgery, bed rest or accident; nerve damage due to spinal cordinjury, autoimmune disease, or infectious disease; glucocorticoid usefor unrelated conditions; sepsis due to infection or other causes;nutrient limitation due to illness or starvation; and space travel. Ageand hormone related bone loss (osteoporosis) are also included.

In yet another embodiment, the treatment of a CRF₂R modulated disorderincludes disorders affecting the heart and circulatory system includingbut not limited to hypertension, congestive heart failure, damage to theheart resulting from heart attack, ischemia reperfusion injury, stroke,migraine, memory loss, Alzheimer's disease, dementia, and the like.

In yet another embodiment, the treatment of a CRF₂R modulated disorderincludes disorder affecting the joints including but not limited toarthritis in particular osteoarthritis and rheumatoid arthritis.

In yet another embodiment, the treatment of a CRF₂R modulated disorderincludes metabolic diseases including obesity and diabetes.

In yet another embodiment, the treatment of a CRF2R modulated disorderincludes: pain reduction; swelling reduction; allergic reactions,allergy; reducing body temperature; suppressing appetite; congestiveheart failure; stress and anxiety; altering undesirably low levels ofadrenocorticotropic hormone (“ACTH”) secretion; controlling appetite,arousal, and cognitive functions; and preventing long term effects ofstress, such as anxiety disorders, anorexia nervosa and melancholicdepression.

The term “treatment” is herein to mean that, at a minimum,administration of a peptide of the present invention that mitigates aCRF₂R modulated disorder in a mammalian subject, preferably in humans.Thus, the term “treatment” includes: preventing a CRF₂R modulateddisorder from occurring in a mammal, particularly when the mammal ispredisposed to acquiring the CRF₂R modulated disorder, but has not yetbeen diagnosed with the disease; inhibiting the CRF₂R modulateddisorder; and/or alleviating or reversing the CRF₂R modulated disorder.Insofar as the methods of the present invention are directed topreventing the CRF₂R modulated disorder, it is understood that the term“prevent” does not require that the CRF₂R modulated disorder becompletely thwarted (see Webster's Ninth Collegiate Dictionary). Rather,as used herein, the term “preventing” refers to the ability of theskilled artisan to identify a population that is susceptible to CRF₂Rmodulated disorders, such that administration of the peptides and kitsof the present invention may occur prior to the onset of the symptoms ofthe CRF₂R modulated disorder. The population that is at risk for aparticular CRF₂R modulated disorder is readily identifiable. Forexample, the population that is at risk for developing musculardystrophy can be determined by identifying mutations in genescharacteristic of the disorder. For example, and previously discussed,Duchenne and Becker dystrophies results from the inheritance of amutation in the dystrophy gene, which is located at the Xp21 locus.Those individuals of a population that possess these mutations are atrisk of developing muscular dystrophy. Thus, the patient population isidentifiable and could receive the administration of a composition orunit dose form of a kit of the present invention before progression ofthe disease. Thus, progression of muscular atrophy or wasting in suchindividuals would be “prevented.”

Nucleic Acid Molecules

The present invention further provides nucleic acid molecules thatencode the peptides of Formula (I) and variants thereof, preferably inisolated form. As used herein, “nucleic acid” is defined as RNA or DNAthat encodes a peptide of the present invention as defined above, or iscomplementary to a nucleic acid sequence encoding such peptides.Specifically contemplated are genomic DNA, cDNA, mRNA and antisensemolecules, as well as nucleic acids based on alternative backbones orincluding alternative bases whether derived from natural sources orsynthesized.

The present invention further provides a fragment of an encoding nucleicacid molecule. As used herein, a fragment of an encoding nucleic acidmolecule refers to a small portion of the entire protein codingsequence. The size of the fragment will be determined by the intendeduse. For example, if the fragment is chosen so as to encode an activeportion of a peptide of the present invention, the fragment will need tobe large enough to encode the functional regions of the peptide.

Fragments of the encoding nucleic acid molecules of the presentinvention (i.e., synthetic oligonucleotides) that are used as probes orspecific primers for the polymerase chain reaction (PCR), or tosynthesize gene sequences encoding peptides of the invention, can easilybe synthesized by chemical techniques, for example, the phosphotriestermethod of Matteucci et al., J. Am. Chem. Soc., 103:3185–3191 (1981) orusing automated synthesis methods. In addition, larger DNA segments canreadily be prepared by well-known methods, such as synthesis of a groupof oligonucleotides that define various modular segments of the gene,followed by ligation of oligonucleotides to build the complete modifiedgene.

The encoding nucleic acid molecules of the present invention may furtherbe modified so as to contain a detectable label for diagnostic and probepurposes. A variety of such labels are known in the art and can readilybe employed with the encoding molecules herein described. Suitablelabels include, but are not limited to, biotin, radiolabeled nucleotidesand the like. A skilled artisan can readily employ any such label toobtain labeled variants of the nucleic acid molecules of the invention.Modifications to the primary structure itself by deletion, addition, oralteration of the amino acids incorporated into the protein sequenceduring translation can be made without destroying the activity of theprotein. Such substitutions or other alterations result in proteinshaving an amino acid sequence encoded by a nucleic acid falling withinthe contemplated scope of the present invention.

Preparation of Peptides or Cell Lines Expressing Peptides

The peptides of the present invention can be prepared for a variety ofuses, including, but not limited to, use as pharmaceutical reagents forthe treatment of CRF₂R modulated disorders. It will be clear to one ofskill in the art that, for certain embodiments of the invention,purified peptides will be most useful, while for other embodiments celllines expressing the peptides will be most useful.

Because the peptides of Formula (I) are short polypeptides, the skilledartisan will recognize that peptides of the present invention may besynthesized by direct synthesis, rather than by recombinant means, usingtechniques well known in the art. See Bodanszky, Principles of PeptideSynthesis, Springer-Verlag, Heidelberg (1984); and such as viasolid-phase synthesis, see, e.g., Merrifield, J. Am. Chem. Soc.,85:2149–54 (1963); Barany et al., Int. J. Peptide Protein Res.,30:705–739 (1987); and U.S. Pat. No. 5,424,398.

For example, the peptides can be synthesized with either an AppliedBiosystem, Inc. (ABI) Model 433 automated synthesizer or a multi-reactorsynthesizer (model Symphony™) from Protein Technology, Inc (PTI). As topeptides synthesized with the ABI synthesizer, all reagents arepurchased from ABI (except piperidine which is purchased from Aldrich).Fmoc amino acids are purchased from ABI (except Fmoc-L-Pyr which ispurchased from Chem-Impek). Rink Amide resins are purchased from NovaChemicals. Standard 0.1 mmole FastMoc chemistry with single coupling isused. The general Fmoc chemistry protocol for SPPS (solid phase peptidesynthesis) includes: 1) cleavage of the Fmoc protection groups withpiperidine; 2) activation of the carboxyl group of amino acids; and 3)coupling the activated amino acids to the amino-terminal of the resinbound peptide chain to form peptide bonds. Amino acids are activatedwith 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU). A dry protected amino acid in a cartridge(1.0 mmol) is dissolved in a solution of HBTU, N,N-diisopropylethylamine(DIEA), and 1-hydroxybenzotriazole (HOBt) in N,N-dimethylformamide (DMF)with additional N-methylpyrrolidone (NMP) added. The activated Fmocamino acid is formed almost instantaneously and the solution istransferred directly to the reaction vessel. The step of Fmocdeprotection is monitored and controlled by conductivity measurement.The peptide chain is built on a Rink Amide resin since the C-terminalamide is needed. The final product is washed extensively with NMP anddichloromethane (DCM).

As to peptides synthesized with the PTI multiple synthesizer, all theFmoc amino acids are purchased from NovaBiochem (except Fmoc-Pyr whichis purchased from Chem-Impex). Standard 0.05 mmole Fmoc synthesisprotocols are used for syntheses. Fmoc amino acids (0.4 mmol) aredissolved in a solution of IBTU (200 mM), N-methylmorpholine (NMM, 0.4M) and N,N-dimethylformamide (DMF) with additional N-methylpyrrolidone(NMP) added. The activated Fmoc amino acid is formed almostinstantaneously and the solution is transferred directly to the reactionvessel. The step of Fmoc deprotection is conducted twice. The peptidechain is built on a Rink Amide resin since the C-terminal amide isneeded. The final synthesis product is washed extensively with NMP anddichloromethane (DCM).

The newly synthesized peptides are deprotected. The resins containingsynthesized peptides are unloaded from the synthesizer and brieflyair-dried. Using 1.5–2.0 ml of the cleavage cocktail (comprising 95%trifluoroacetic acid (TFA), 2.5% ethanodithiol, 2.5% thioanisol, 2.5%phenol (W/V) in water) for 4 hours at room temperature, the peptides arecleaved off the resin and at the same time, the side chain protectiongroups [O-t-butyl (OtBu) for Asp, Glu, Tyr, Thr and Ser;Pentamethylchroman-6-sulfonyl (Pmc) for Arg, t-butoxycarbonyl (Boc) forTrp and Lys; trityl (Trt) for His, Asn and Gln] are removed under thedeprotection condition. The cleavage solution is separated from theresin by filtration. The filtrate is then diluted with 15 ml of water.Six rounds of ether extraction are performed to clean the peptideproduct. The peptide is lyophilized and stored at −20° C. beforepurification.

The deprotected peptides are purified and characterized. The peptidepowder is dissolved in 50% acetic acid solution and injected onto aVydac 1.0 cm I.D. 25 cm length C-8 column with 5 μm particle size, and300 Å pore size for purification. A Beckman System Gold high performanceliquid chromatography (HPLC) system with dual wavelength (220 nm and 280nm) ultraviolet detector is used. A linear gradient of acetonitrile isprogrammed and introduced to the column to separate the peptide productfrom other substances. The elute is collected by a Pharmacia fractioncollector, and the individual separation fractions were subjected toboth analytical HPLC and (matrix assisted laser desorption ionizationtime of flight mass spectroscopy) MALDI-TOF MS for characterization toensure the identity and purity.

The use of recombinant DNA technology in the preparation of thepeptides, or of cell lines expressing these peptides, is alsocontemplated. Such recombinant methods are well known in the art.Methods for generating rDNA molecules are well known in the art, forexample, see Sambrook et al., Molecular Cloning—A Laboratory Manual,Cold Spring Harbor Laboratory Press (1989). To express recombinantpeptides of the present invention, an expression vector that comprises anucleic acid which encodes the polypeptide of interest under the controlof one or more regulatory elements, is prepared. The sequence of nucleicacids encoding the peptides of the present invention can be deduced fromthe peptide sequences discussed or claimed herein.

By methods well known in the art, the isolated nucleic acid moleculeencoding the peptide of interest may be ligated into a suitableexpression vector. The host-expression vector systems that may be usedfor purposes of the invention include, but are not limited to:microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA, or cosmid DNAexpression vectors containing nucleotide sequences encoding the peptidesof the present invention; yeast (e.g., Saccharomyces, Pichia)transformed with recombinant yeast expression vectors containingnucleotide sequences encoding the peptides of the present invention;insect cell systems infected with recombinant virus expression vectors(e.g., baculovirus) containing nucleotide sequences encoding thepeptides of the present invention; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,tobacco mosaic virus) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasmid) containing nucleotide sequences encoding thepeptides of the present invention; or mammalian cell systems (e.g., COS,CHO, HEK293, NIH3T3) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., retrovirusLTR) and also containing nucleotide sequences encoding the peptides ofthe present invention.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the peptidebeing expressed. For example, when a large quantity of such protein isneeded, vectors which direct the expression of high levels of proteinproducts are desirable. One skilled in the art is able to generate suchvector constructs and purify the proteins by a variety of methodologiesincluding selective purification technologies such as fusion proteinselective columns and antibody columns, and non-selective purificationtechnologies.

In an insect protein expression system, the baculovirus A. californicanuclear polyhedrosis virus (AcNPV), is used as a vector to expressforeign genes in S. frugiperda cells. In this case, nucleotide sequencesencoding the peptides of the present invention are cloned intonon-essential regions of the virus and placed under the control of anAcNPV promoter. The recombinant viruses are then used to infect cells inwhich the inserted gene is expressed and the protein is purified by oneof many techniques known to one skilled in the art.

In mammalian host cells, a number of viral-based expression systems maybe utilized. Utilization of these expression systems often requires thecreation of specific initiation signals in the vectors for efficienttranslation of the inserted nucleotide sequences. This is particularlyimportant if a portion of the nucleotide sequence used does not containthe endogenous initiation signal. The placement of this initiationsignal, in frame with the coding region of the inserted nucleotidesequence, as well as the addition of transcription and translationenhancing elements and the purification of the recombinant protein, areachieved by one of many methodologies known to one skilled in the art.Also important in mammalian host cells is the selection of anappropriate cell type which is capable of the necessary posttranslational modifications of the recombinant protein. Suchmodifications, for example, cleavage, phosphorylation, glycosylation,acetylation, etc., require the selection of the appropriate host cellwhich contains the modifying enzymes. Such host cells include, but arenot limited to, CHO, HEK293, NIH3T3, COS, etc. and are known by thoseskilled in the art.

For long term, high expression of recombinant proteins, stableexpression is preferred. For example, cell lines that stably expresspeptides of the present invention may be engineered. One of skill in theart, following known methods such as electroporation, calcium phosphatetransfection, or liposome-mediated transfection, can generate a cellline that stably expresses the peptides of the present invention. Thisis usually accomplished by transfecting cells using expression vectorswhich contain appropriate expression control elements (e.g., promotersequences, enhancer sequences, transcriptional termination sequences,polyadenylation sites, translational start sites, etc.), a selectablemarker, and the gene of interest. The selectable marker may either becontained within the same vector, as the gene of interest, or on aseparate vector, which is co-transfected with the peptide encodingsequence-containing vector. The selectable marker in the expressionvector may confer resistance to the selection and allows cells to stablyintegrate the vector into their chromosomes and to grow to form fociwhich in turn can be cloned and expanded into cell lines. Alternatively,the expression vector may allow selection of the cell expressing theselectable marker utilizing a physical attribute of the marker, i.e.,expression of Green Fluorescent Protein (GFP) allows for selection ofcells expressing the marker using fluorescence activated cell sorting(FACS) analysis.

One of skill in the art is able to select an appropriate cell type fortransfection in order to allow for selection of cells into which thesequence of interest has been successfully integrated. For example,where the selectable marker is herpes simplex virus thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase or adeninephosphoribosyltransferase, the appropriate cell type would be tk-,hgprt- or aprt-cells, respectively. Or, normal cells can be used wherethe selectable marker is dhfr, gpt, neo or hygro which confer resistanceto methotrexate, mycophenolic acid, G-418 or hygromycin, respectively.

Preparation of Antibodies

Antibodies that selectively recognize one or more epitopes of thepeptides of the present invention are also encompassed by the invention.Such antibodies include, e.g., polyclonal antibodies, monoclonalantibodies, chimeric antibodies, human antibodies, single chainantibodies, Fab fragments, F(ab′)₂ fragments, molecules produced using aFab expression library, human antibodies (polyclonal or monoclonal)produced in transgenic mice and epitope binding fragments of any of theabove.

The antibodies can be utilized in conjunction with gene therapytechniques to evaluate, for example, the expression of the peptides ofthe present invention either in cells or directly in patient tissues inwhich these genes have been introduced.

For the production of antibodies, a variety of host animals may beimmunized by injection with peptides of the present invention,anti-peptide antibody, anti-peptide analog antibody, or immunogenicfragments thereof by methods well known in the art. For preparation ofan anti-idiotype antibody the immunogen is an anti-peptide antibody oranti-peptide analog antibody. Production of anti-idiotype antibodies isdescribed, for example, in U.S. Pat. No. 4,699,880. Suitable hostanimals include, but are not limited to, rabbits, mice, goats, sheep andhorses. Immunization techniques are well known in the art. Polyclonalantibodies can be purified from the serum of the immunized animals, ormonoclonal antibodies can be generated by methods that are well known inthe art. These techniques include, but are not limited to, thewell-known hybridoma techniques of Kohler and Milstein, human B-cellhybridoma techniques, and the EBV hybridoma technology. Monoclonalantibodies may be of any immunoglobulin class, including IgG, IgE, IgM,IgA, and IgD containing either kappa or lambda light chains. Techniquesof producing and using chimeric antibodies are known in the art, and aredescribed in, for example, U.S. Pat. Nos. 5,807,715; 4,816,397;4,816,567; 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370; and5,824,307.

Assays Determining CRF₂R Selectivity

The pharmacological activity and selectivity of the peptides of presentinvention can be determined using published test procedures. See, e.g,U.S. pat. appl. Ser. No. 09/799978. Because CRF₂R and CRF₁R arehomologous proteins, it is expected that a certain proportion ofagonists for CRF₂R will also function as agonists of CRF₁R. As discussedabove, activation of CRF₁R induces activation of the HPA axis sinceincreased corticosteroid production leads to skeletal muscle atrophy. Inmost cases in which an increase in muscle mass or function is desired,it is not desirable to activate the HPA axis. When selecting a peptideuseful for the treatment of a CRF₂R modulated disorder, which is notrelated to muscular dystrophy, it is preferable that the peptide beselective for CRF₂R. Preferably the peptide exhibits 10-fold selectivityfor CRF₂R versus CRF₁R (i.e., 10-fold more active against CRF₂R thanagainst CRF₁R), more preferably 100-fold selectivity and most preferably1000-fold or greater selectivity. As published studies have demonstrateda benefit of corticosteroid therapy in the treatment of musculardystrophies, it may be beneficial that a CRF₂R agonist retain some levelof CRF₁R agonism when used to treat muscular dystrophies. Thus, for thetreatment of muscular dystrophies, a peptide of lower selectivity thatactivates the CRF₂R as well as the CRF₁R, over a similar concentrationrange, is preferred. Preferably the peptide is 100-fold selective forCRF₂R versus CRF₁R, more preferably 10-fold selective and mostpreferably not selective for CRF₂R versus CRF₁R (i.e., the activity ofthe candidate compound is substantially similar for CRF₂R and CRF₁R).Also, in this case, it may be more preferable that the peptide is fullagonist for CRF₂R while being a partial agonist for, CRF₁R. Such apeptide would therefore have a built-in limit to the maximum degree ofcortisol elevation and potential for muscle atrophy, while theanti-atrophy effect modulated through the CRF₂R could be enhanced byincreasing the dose. One of skill in the art would be able to readilydetermine whether a peptide is a full or partial agonist of the CRF₁R orCRF₂R using methods known in the art.

Because it is desirable to discriminate binding between CRF₂R, ascompared with CRF₁R, the assays described above may be conducted using acell, or membrane from a cell, which expresses only CRF₂R or the assayscan be conducted with a recombinant source of CRF₂R. Cells expressingboth forms of CRFR may be modified using homologous recombination toinactivate or otherwise disable the CRF₁R gene. Alternatively, if thesource of CRFR contains more than one CRFR type, the background signalproduced by the receptor which is not of interest must be subtractedfrom the signal obtained in the assay. The background response can bedetermined by a number of methods, including elimination of the signalfrom the CRFR which is not of interest by use of antisense, antibodiesor selective antagonists. Known antagonists of CRFRs include, but arenot limited to, antalarmin (CRF₁R selective), antisauvagine-30 (CRF₂Rselective) and astressin (nonselective for CRF₁R/CRF₂R).

To determine whether a peptide activates CRF₂R and/or CRF₁R, the assaysare typically cell-based; however, cell-free assays are known which areable to differentiate agonist and antagonist binding as described above.Cell-based assays include the steps of contacting cells which expressCRF₁R or CRF₂R with a peptide of the present invention or control andmeasuring activation of the CRFR by measuring the expression or activityof components of the CRFR signal transduction pathways.

As described in the background section above, CRFRs appear to couplethrough several different pathways including G_(αs), G_(αq) or G_(αi),depending upon the cell type. It is thought that agonist activation ofCRFR allows the receptor to signal via any of these pathways, providedthat the necessary pathway components are present in the particular celltype. Thus, to assay a particular peptide of the present invention forCRFR activation, an assay can use any of the signal transductionpathways as the readout even if the relevant cell type for treatment, invivo, couples CRFR to skeletal muscle atrophy via a different pathway.One of ordinary skill in the art would recognize that an assay would beeffective for identifying useful peptide agonists independent of thepathway by which receptor activation was measured. Assays for measuringactivation of these signaling pathways are known in the art.

For example, after contact with a peptide of the present invention,lysates of the cells can be prepared and assayed for induction of cAMP.cAMP is induced in response to G_(αs) activation. Because G_(αs) isactivated by receptors other than CRFR and because a test peptide may beexerting its effect through CRFRs or by another mechanism, two controlcomparisons are relevant for determining whether the peptide increaseslevels of cAMP via activation of a CRFR. One control compares the cAMPlevel of cells contacted with the peptide and the cAMP level of cellscontacted with a control compound (i.e., the vehicle in which thepeptide is dissolved). If the peptide increases cAMP levels relative tothe control compound this indicates that the peptide is increasing cAMPby some mechanism. The other control compares the cAMP levels of a CRFRexpressing cell line and a cell line that is essentially the same exceptthat it does not express the CRFR, where both of the cell lines havebeen treated with the peptide. If the peptide elevates cAMP levels inthe CRFR expressing cell line relative to the cell line that does notexpress CRFRs, this is an indication that the peptide elevates cAMP viaactivation of the CRFRs.

In one example, cAMP induction is measured with the use of DNAconstructs containing the cAMP responsive element linked to any of avariety of reporter genes can be introduced into cells expressing CRFRs.Such reporter genes include, but are not limited to, chloramphenicolacetyltransferase (CAT), luciferase, glucuronide synthetase, growthhormone, fluorescent proteins (e.g., Green Fluorescent Protein), oralkaline phosphatase. Following exposure of the cells to the peptide,the level of reporter gene expression can be quantitated to determinethe peptide's ability to increase cAMP levels and thus determine thepeptide's ability to activate the CRFR.

The cells useful in this assay are the same as for the CRFR bindingassay described above, except that cells utilized in the activationassays preferably express a functional receptor which gives astatistically significant response to CRF or one or more CRF analog. Inaddition to using cells expressing full length CRFRs, cells can beengineered which express CRFRs containing the ligand binding domain ofthe receptor coupled to, or physically modified to contain, reporterelements or to interact with signaling proteins. For example, a wildtype CRFR or CRFR fragment can be fused to a G-protein resulting inactivation of the fused G-protein upon agonist binding to the CRFRportion of the fusion protein. Siefert, R. et al., Trends Pharmacol.Sci., 20: 383–389 (1999). The cells should also preferably possess anumber of characteristics, depending on the readout, to maximize theinductive response by CRF or the CRF analog, for example, for detectinga strong induction of a CRE reporter gene; (a) a low natural level ofcAMP; (b) G proteins capable of interacting with CRFRs; (c) a high levelof adenylyl cyclase; (d) a high level of protein kinase A; (e) a lowlevel of phosphodiesterases; and (f) a high level of cAMP responseelement binding protein would be advantageous. To increase the responseto CRF or a CRF analog, host cells could be engineered to express agreater amount of favorable factors or a lesser amount of unfavorablefactors. In addition, alternative pathways for induction of the CREreporter could be eliminated to reduce basal levels.

Assays to Determine Pharmacological Activity

The pharmacological activity of the peptides of present invention can bedetermined using published test procedures. For example, models ofskeletal muscle atrophy or hypertrophy include both in vitro cellculture models and in vivo animal models of skeletal muscle atrophy.

In vitro models of skeletal muscle atrophy are known in the art. Suchmodels are described, for example, in Vandenburgh, H. H., In Vitro,24:609–619 (1988), Vandenburgh, H. H. et al., J. Biomechanics, 24 Suppl1:91–99 (1991), Vandenburgh, H. H et al., In Vitro Cell. Dev. Biol.,24(3):166–174 (1988), Chromiak, J. A., et al., In Vitro Cell. Dev. Biol.Anim., 34(9):694–703 (1998), Shansky, J., et al., In Vitro Cell. Dev.Biol. Anim., 33(9):659–661 (1997), Perrone, C. E. et al., J. Biol.Chem., 270(5):2099–2106 (1995), Chromiac, J. A. and Vandenburgh, H. H.,J. Cell. Physiol., 159(3):407–414 (1994), and Vandenburgh, H. H. andKarlisch, P., In Vitro Cell. Dev. Biol., 25(7):607–616 (1989).

A variety of animal models for skeletal muscle atrophy are known in theart, such as those described in the following references: Herbison, G.J., et al. Arch. Phys. Med. Rehabil., 60:401–404 (1979), Appell, H-J.Sports Medicine 10:42–58 (1990), Hasselgren, P-O. and Fischer, J. E.World J. Surg., 22:203–208 (1998), Agbenyega, E. T. and Wareham, A. C.Comp. Biochem. Physiol., 102A:141–145 (1992), Thomason, D. B. and Booth,F. W. J. Appl. Physiol., 68:1–12 (1990), Fitts, R. H., et al. J. Appl.Physiol., 60:1946–1953 (1986), Bramanti, P., et al. Int. J. Anat.Embryol. 103:45–64 (1998), Cartee, G. D. J. Gerontol. A Biol. Sci. Med.Sci., 50:137–141 (1995), Cork, L. C., et al. Prog. Clin. Biol. Res.,229:241–269 (1987), Booth, F. W. and Gollnick, P. D. Med. Sci. SportsExerc., 15:415–420 (1983), Bloomfield, S. A. Med. Sci. Sports Exerc.,29:197–206 (1997). Preferred animals for these models are mice and rats.These models include, for example, models of disuse-induced atrophy suchas casting or otherwise immobilizing limbs, hind limb suspension,complete animal immobilization, and reduced gravity situations. Modelsof nerve damage induced atrophy include, for example, nerve crush,removal of sections of nerves which innervate specific muscles, toxinapplication to nerves and infection of nerves with viral, bacterial oreukaryotic infectious agents. Models of glucocorticoid-induced atrophyinclude application of atrophy-inducing doses of exogenousglucocorticoid to animals, and stimulation of endogenous corticosteroidproduction, for example, by application of hormones that activate thehypothalamus-pituitary-adrenal (HPA) axis. Models of sepsis-inducedatrophy include, for example, inoculation with sepsis-inducing organismssuch as bacteria, treatment of the animal with immune-activatingcompounds such as bacterial cell wall extract or endotoxin, and punctureof intestinal walls. Models of cachexia-induced atrophy include, forexample, inoculation of an animal with tumorigenic cells with cachexiaforming potential, infection of an animal with infectious agents (suchas viruses which cause AIDS) which result in cachexia and treatment ofan animal with hormones or cytokines such as CNTF, TNF, IL-6, IL-1, etc.which induce cachexia. Models of heart failure-induced atrophy includethe manipulation of an animal so that heart failure occurs withconcomitant skeletal muscle atrophy. Neurodegenerative disease-inducedatrophy models include autoimmune animal models such as those resultingfrom immunization of an animal with neuronal components. Musculardystrophy-induced models of atrophy include natural or man-madegenetically induced models of muscular dystrophy such as the mutation ofthe dystrophin gene which occurs in the Mdx mouse.

Animal models of skeletal muscle hypertrophy include, for example,models of increased limb muscle use due to inactivation of the opposinglimb, reweighing following a disuse atrophy inducing event,reutilization of a muscle which atrophied because of transient nervedamage, increased use of selective muscles due to inactivation of asynergistic muscle (e.g., compensatory hypertrophy), increased muscleutilization due to increased load placed on the muscle and hypertrophyresulting from removal of the glucocorticoid afterglucocorticoid-induced atrophy. Preferred animal atrophy models includethe sciatic nerve denervation atrophy model, glucocorticoid-inducedatrophy model, and the leg casting disuse atrophy model that aredescribed in further detail below.

The sciatic nerve denervation atrophy model involves anesthetizing theanimal followed by the surgical removal of a short segment of either theright or left sciatic nerve, e.g., in mice the sciatic nerve is isolatedapproximately at the midpoint along the femur and a 3–5 mm segment isremoved. This denervates the lower hind limb musculature resulting inatrophy of these muscles. Typically, innervation to the biceps femorisis left intact to provide satisfactory motion of the knee for virtuallynormal ambulation. Typically, in untreated animals, muscle mass of thedenervated muscles is reduced 30–50% ten days following denervation.Following denervation, test peptides are administered e.g., by injectionor by continuous infusion, e.g., via implantation of an osmotic minipump(e.g., Alzet, Palo Alto, Calif.), to determine their effect ondenervation induced skeletal muscle atrophy. At various times followingdenervation, the animals are euthanized and lower leg muscles aredissected rapidly from both the denervated and nondenervated legs, themuscles, cleaned of tendons and connective tissue, are weighed. Theextent of atrophy in the affected muscles is analyzed, for example, bymeasuring muscle mass, muscle cross-sectional area, myofibercross-sectional area or contractile protein content.

The glucocorticoid-induced atrophy model involves the administration ofa glucocorticoid to the test animal, e.g., 1.2 mg/kg/day ofdexamethasone in the drinking water. Typically, in untreated animals,skeletal muscle mass is reduced 30–50% following ten days ofdexamethasone administration. Concomitantly with, or followingglucocorticoid administration, test peptides are administered e.g., byinjection or by continuous infusion to determine their effect onglucocorticoid-induced skeletal muscle atrophy. At various timesfollowing glucocorticoid administration, the extent of atrophy in theaffected muscles is analyzed as described above for the denervationmodel.

The leg casting disuse atrophy model involves casting one hind leg of ananimal from the knee down through the foot. Typically, muscle mass isreduced 20–40% after ten days of casting. Following casting, testpeptides are administered by injection or by continuous infusion viaimplantation of an osmotic minipump (e.g., Alzet, Palo Alto, Calif.) todetermine their effect on leg casting induced skeletal muscle atrophy.At various times following leg casting, the extent of atrophy in theaffected muscles is analyzed as described above for the denervationmodel.

Bone activity of the subject peptides can be conveniently demonstratedusing an assay designed to test the ability of the subject compounds toincrease bone volume, mass, or density. An example of such assays is theovariectomized rat assay.

In the ovariectomized rat assay, six-month old rats are ovariectomized,aged 2 months, and then dosed once a day subcutaneously with a testcompound. Upon completion of the study, bone mass and/or density can bemeasured by dual energy x-ray absorptometry (DXA) or peripheralquantitative computed tomography (pQCT), or micro computed tomography(mCT). Alternatively, static and dynamic histomorphometry can be used tomeasure the increase in bone volume or formation.

Compositions

Another aspect of this invention is compositions which comprise: (a) asafe and effective amount of a peptide of the present invention; and (b)a pharmaceutically-acceptable carrier. Standard pharmaceuticalformulation techniques are used, such as those disclosed in Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., latestedition.

A “safe and effective amount” means an amount of the peptide of theinvention sufficient to significantly induce a positive modification inthe condition to be treated, but low enough to avoid serious sideeffects (such as toxicity, irritation, or allergic response) in ananimal, preferably a mammal, more preferably a human subject, in needthereof, commensurate with a reasonable benefit/risk ratio when used inthe manner of this invention. The specific “safe and effective amount”will, obviously, vary with such factors as the particular conditionbeing treated, the physical condition of the subject, the duration oftreatment, the nature of concurrent therapy (if any), the specificdosage form to be used, the carrier employed, the solubility of thepeptide therein, and the dosage regimen desired for the composition. Oneskilled in the art may use the following teachings to determine a “safeand effective amount” in accordance with the present invention. SpilkerB., Guide to Clinical Studies and Developing Protocols, Raven PressBooks, Ltd., New York, 1984, pp. 7–13, 54–60; Spilker B., Guide toClinical Trials, Raven Press, Ltd., New York, 1991, pp. 93–101; CraigC., and R. Stitzel, eds., Modern Pharmacology, 2d ed., Little, Brown andCo., Boston, 1986, pp. 127–33; T. Speight, ed., Avery's Drug Treatment:Principles and Practice of Clinical Pharmacology and Therapeutics, 3ded., Williams and Wilkins, Baltimore, 1987, pp. 50–56; R. Tallarida, R.Raffa and P. McGonigle, Principles in General Pharmacology,Springer-Verlag, New York, 1988, pp. 18–20.

In addition to the subject peptide, the compositions of the subjectinvention contain a pharmaceutically acceptable carrier. The term“pharmaceutically-acceptable carrier,” as used herein, means one or morecompatible solid or liquid filler diluents or encapsulating substanceswhich are suitable for administration to an animal, preferably a mammal,more preferably a human. The term “compatible”, as used herein, meansthat the components of the composition are capable of being commingledwith the subject peptide, and with each other, in a manner such thatthere is no interaction that would substantially reduce thepharmaceutical efficacy of the composition under ordinary usesituations. Pharmaceutically-acceptable carriers must, of course, be ofsufficiently high purity and sufficiently low toxicity to render themsuitable for administration to the animal, preferably a mammal, morepreferably a human being treated.

Some examples of substances which can serve aspharmaceutically-acceptable carriers or components thereof are: sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powderedtragacanth; malt; gelatin; talc; solid lubricants, such as stearic acidand magnesium stearate; calcium sulfate; vegetable oils, such as peanutoil, cottonseed oil, sesame oil, olive oil, corn oil and oil oftheobroma; polyols such as propylene glycol, glycerine, sorbitol,mannitol, and polyethylene glycol; alginic acid; emulsifiers, such asthe Tweens®; wetting agents, such sodium lauryl sulfate; coloringagents; flavoring agents; tableting agents, stabilizers; antioxidants;preservatives; pyrogen-free water; isotonic saline; and phosphate buffersolutions.

The choice of a pharmaceutically-acceptable carrier to be used inconjunction with the subject compound is basically determined by the waythe peptide is to be administered.

If the subject peptide is to be injected, the preferredpharmaceutically-acceptable carrier is sterile, physiological saline,with a blood-compatible colloidal suspending agent, the pH of which hasbeen adjusted to about 7.4.

In particular, pharmaceutically-acceptable carriers for systemicadministration include sugars, starches, cellulose and its derivatives,malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils,polyols, alginic acid, phosphate buffer solutions, emulsifiers, isotonicsaline, and pyrogen-free water. Preferred carriers for parenteraladministration include propylene glycol, ethyl oleate, pyrrolidone,ethanol, and sesame oil. Preferably, the pharmaceutically-acceptablecarrier, in compositions for parenteral administration, comprises atleast about 90% by weight of the total composition.

The compositions of this invention are preferably provided in unitdosage form. As used herein, a “unit dosage form” is a composition ofthis invention containing an amount of a Formula (I) peptide that issuitable for administration to an animal, preferably a mammal, morepreferably a human subject, in a single dose, according to good medicalpractice. These compositions preferably contain from about 0.1 mg(milligrams) to about 1000 mg, more preferably from about 0.5 mg toabout 500 mg, more preferably from about 1 mg to about 30 mg, of apeptide of Formula (I).

The compositions of this invention may be in any of a variety of forms,suitable, for example, for oral, rectal, topical, nasal, ocular orparenteral administration. Depending upon the particular route ofadministration desired, a variety of pharmaceutically-acceptablecarriers well-known in the art may be used. These include solid orliquid fillers, diluents, hydrotropes, surface-active agents, andencapsulating substances. Optional pharmaceutically-active materials maybe included, which do not substantially interfere with the CRF₂R agonistactivity of the peptides of Formula (I). The amount of carrier employedin conjunction with the Formula (I) peptide is sufficient to provide apractical quantity of material for administration per unit dose of theFormula (I) peptide. Techniques and compositions for making dosage formsuseful in the methods of this invention are described in the followingreferences,: Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes,editors, 1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets(1981); and Ansel, Introduction to Pharmaceutical Dosage Forms 2dEdition (1976).

Various oral dosage forms can be used, including such solid forms astablets, capsules, granules and bulk powders. These oral forms comprisea safe and effective amount, usually at least about 5%, and preferablyfrom about 25% to about 50%, of the peptide. Tablets can be compressed,tablet triturates, enteric-coated, sugar-coated, film-coated, ormultiple-compressed, containing suitable binders, lubricants, diluents,disintegrating agents, coloring agents, flavoring agents, flow-inducingagents, and melting agents. Liquid oral dosage forms include aqueoussolutions, emulsions, suspensions, solutions and/or suspensionsreconstituted from non-effervescent granules, and effervescentpreparations reconstituted from effervescent granules, and containingsuitable solvents, preservatives, emulsifying agents, suspending agents,diluents, sweeteners, melting agents, coloring agents and flavoringagents.

The pharmaceutically-acceptable carrier suitable for the preparation ofunit dosage forms for peroral administration are well-known in the art.Tablets typically comprise conventional pharmaceutically-compatibleadjuvants as inert diluents, such as calcium carbonate, sodiumcarbonate, mannitol, lactose and cellulose; binders such as starch,gelatin and sucrose; disintegrants such as starch, alginic acid andcroscarmelose; lubricants such as magnesium stearate, stearic acid andtalc. Glidants such as silicon dioxide can be used to improve flowcharacteristics of the powder mixture. Coloring agents, such as the FD&Cdyes, can be added for appearance. Sweeteners and flavoring agents, suchas aspartame, saccharin, menthol, peppermint, and fruit flavors, areuseful adjuvants for chewable tablets. Capsules typically comprise oneor more solid diluents disclosed above. The selection of carriercomponents depends on secondary considerations like taste, cost, andshelf stability, which are not critical for the purposes of the subjectinvention, and can be readily made by a person skilled in the art. Ingeneral, the formulation will include the peptide, and inert ingredientswhich allow for protection against the stomach environment, and releaseof the biologically active material in the intestine.

The peptide of Formula (I) may be chemically modified so that oraldelivery of the derivative is efficacious. Generally, the chemicalmodification contemplated is the attachment of at least one moiety tothe protein molecule itself, where said moiety permits (a) inhibition ofproteolysis; and (b) uptake into the blood stream from the stomach orintestine. Also desired is the increase in overall stability of theprotein and increase in circulation time in the body. Examples of suchmoieties include: polyethylene glycol, copolymers of ethylene glycol andpropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinyl pyrrolidone and polyproline. Newmark et al., J. Appl.Biochem., 4:185–189 (1982). Other polymers that could be used arepoly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred forpharmaceutical usage, as indicated above, are polyethylene glycolmoieties.

The location of release may be the stomach, the small intestine (theduodenum, the jejunem, or the ileum), or the large intestine. Oneskilled in the art has available formulations which will not dissolve inthe stomach, yet will release the material in the duodenum or elsewherein the intestine. Preferably, the release will avoid the deleteriouseffects of the stomach environment, either by protection of the peptide(or variant) or by release of the biologically active material beyondthe stomach environment, such as in the intestine.

To ensure full gastric resistance, a coating impermeable to at least pH5.0 is preferred. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

Peroral compositions also include liquid solutions, emulsions,suspensions, and the like. The pharmaceutically-acceptable carrierssuitable for preparation of such compositions are well known in the art.Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include methyl cellulose, sodium carboxymethylcellulose, Avicel® RC-591, tragacanth and sodium alginate; typicalwetting agents include lecithin and polysorbate 80; and typicalpreservatives include methyl paraben and sodium benzoate. Peroral liquidcompositions may also contain one or more components such as sweeteners,flavoring agents and colorants disclosed above.

Compositions of the subject invention may optionally include otheractive agents. Non-limiting examples of active agents are listed in WO99/15210.

Other compositions useful for attaining systemic delivery of the subjectcompounds include sublingual, buccal, suppository, nasal and pulmonarydosage forms. Such compositions typically comprise one or more ofsoluble filler substances such as sucrose, sorbitol and mannitol; andbinders such as acacia, microcrystalline cellulose, carboxymethylcellulose and hydroxypropyl methyl cellulose. Glidants, lubricants,sweeteners, colorants, antioxidants and flavoring agents disclosed abovemay also be included.

The compositions of this invention can also be administered topically toa subject, e.g., by the direct laying on or spreading of the compositionon the epidermal or epithelial tissue of the subject, or transdermallyvia a “patch.” An example of a suitable patch applicator is described inU.S. patent application Ser. No. 10/054113. Such compositions include,for example, lotions, creams, solutions, gels and solids. These topicalcompositions preferably comprise a safe and effective amount, usually atleast about 0.1%, and preferably from about 1% to about 5%, of thepeptide. Suitable carriers for topical administration preferably remainin place on the skin as a continuous film, and resist being removed byperspiration or immersion in water. Generally, the carrier is organic innature and capable of having dispersed or dissolved therein the peptide.The carrier may include pharmaceutically-acceptable emollients,emulsifiers, thickening agents, solvents and the like.

Methods of Administration

This invention also provides methods of treating CRF₂R modulateddisorders in a human or other animal subject, by administering a safeand effective amount of a peptide to said subject. The methods of theinvention are useful in preventing or treating disorders describedabove.

Compositions of this invention can be administered topically orsystemically. Systemic application includes any method of introducing apeptide of Formula (I) into the tissues of the body, e.g.,intra-articular (especially in treatment of rheumatoid arthritis),intrathecal, epidural, intramuscular, transdermal, intravenous,intraperitoneal, subcutaneous, nasal, pulmonary, sublingual, rectal, andoral administration.

The specific dosage of the peptide to be administered, as well as theduration of treatment, and whether the treatment is topical or systemicare interdependent. The dosage and treatment regimen will also dependupon such factors as the specific peptide used, the treatmentindication, the ability of the peptide to reach minimum inhibitoryconcentrations at the site of the tissue in need of treatment, thepersonal attributes of the subject (such as weight), compliance with thetreatment regimen, and the presence and severity of any side effects ofthe treatment.

Topical administration can be used to deliver the peptide systemically,or to treat a subject locally. The amounts of the peptide to betopically administered depends upon such factors as skin sensitivity,type and location of the tissue to be treated, the composition andcarrier (if any) to be administered, the particular peptide to beadministered, as well as the particular disorder to be treated and theextent to which systemic (as distinguished from local) effects aredesired.

The peptides of the present invention can be targeted to specificlocations where treatment is need by using targeting ligands. Forexample, to focus a peptide to treat muscular dystrophy, the peptide isconjugated to an antibody or fragment thereof which is immunoreactivewith a skeletal muscle marker as is generally understood in the art. Thetargeting ligand can also be a ligand suitable for a receptor which ispresent on skeletal muscle. Any targeting ligand which specificallyreacts with a marker for the intended target tissue can be used. Methodsfor coupling the invention compound to the targeting ligand are wellknown and are similar to those described below for coupling to carrier.

A peptide of Formula (I) may be administered via a controlled release.For example, the peptide may be administered using intravenous infusion,an implantable osmotic pump, a transdermal patch, liposomes,subcutaneous depot injection containing a biodegradable material, orother modes of administration. In one embodiment, a pump may be usedLanger et al., eds., Medical Applications of Controlled Release, CRCPres., Boca Raton, Fla. (1974); Sefton, CRC Crit. Ref Biomed. Eng.,14:201 (1987); Buchwald et al., Surgery, 88:507 (1980); Saudek et al.,N. Engl. J. Med., 321:574 (1989). In another embodiment, polymericmaterials can be used. Langer, 1974, supra; Sefton, 1987, supra; Smolenet al., eds., Controlled Drug Bioavailability, Drug Product Design andPerformance, Wiley, N.Y. (1984); Ranger et al., J. Macromol. Sci. Rev.Macromol. Chem., 23:61 (1983); see also Levy et al., Science, 228:190(1985); During et al., Ann. Neurol., 25:351 (1989); Howard et al., J.Neurosurg., 71:105 (1989). In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target thusrequiring only a fraction of the systemic dose. See. e.g., Goodson, inMedical Applications of Controlled Release, vol. 2, pp. 115–138 (1984).In yet another embodiment, a polymer-based drug-delivery system whereindrugs are delivered from polymer or lipid systems. These systems delivera drug by three general mechanisms: (1) diffusion of the drug speciesfrom or through the system; (2) a chemical or enzymatic reaction leadingto degradation of the system, or cleavage of the drug from the system;and (3) solvent activation, either through osmosis or swelling of thesystem. Suitable systems are described in review articles: Langer,Robert, “Drug delivery and targeting,” Nature: 392 (Supp):5–10 (1996);Kumar, Majeti N. V., “Nano and Microparticles as Controlled DrugDelivery Devices,” J Pharm Pharmaceut Sci, 3(2):234–258 (2000);Brannon-Peppas, “Polymers in Controlled Drug Delivery,” Medical Plasticsand Biomaterials, (November 1997). See also, Langer, 1990, supra; Treatet al., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353–365 (1989);Langer, Science, 249:1527–1533 (1990). Suitable systems may include:Atrigel™ drug delivery system from Atrix Labs; DepoFoam™ from SkyPharma;polyethylene glycol-based hydrogels from Infimed Therapeutics, Inc.;ReGel™, SQZGel™ oral, HySolv™ and ReSolv™ solubilizing drug-deliverysystems from MacroMed; ProGelz™ from ProGelz' Products; and ProLease™injectable from Alkermes.

In all of the foregoing, of course, the peptides of the invention can beadministered alone or as mixtures, and the compositions may furtherinclude additional drugs or excipients as appropriate for theindication.

Gene Therapy

Expression vectors may be used to introduce the nucleic acids of theinvention into a cell as part of gene therapy. Such vectors generallyhave convenient restriction sites located near the promoter sequence toprovide for the insertion of nucleic acid sequences. Transcriptioncassettes may be prepared comprising a transcription initiation region,the target gene or fragment thereof, and a transcriptional terminationregion. The transcription cassettes may be introduced into a variety ofvectors, e.g., plasmid, retrovirus, lentivirus, adenovirus and the like,where the vectors are able to transiently or stably be maintained in thecells, usually for a period of at least about one day, more usually fora period of at least about several days to several weeks.

The proteins and nucleic acids of the invention may be introduced intotissues or host cells by any number of routes, including viralinfection, microinjection, or fusion of vesicles. Jet injection may alsobe used for intramuscular administration, as described by Furth et al.,Anal. Biochem., 205:365–368 (1992). The DNA may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature. See, e.g., Tang etal., Nature 356:152–154 (1992), where gold microprojectiles are coatedwith DNA, then bombarded into skin cells.

Kits

The present invention includes a kit for preventing or treating a CRF₂Rmodulated disorder comprising: (a) a peptide of Formula (I) in a unitdose form; and (b) usage instructions. Such a kit preferably includes anumber of unit dosages. Such kits can include a card having dosagesoriented in the order of their intended use. An example of such a kit isa “blister pack.” Blister packs are well known in the packaging industryand are widely used for packaging pharmaceutical unit dosage forms. Ifdesired, a memory aid can be provided, for example in the form ofnumbers, letters, or other markings or with a calendar insert,designating the days in the treatment schedule in which the dosages canbe administered. Example of a kit is described in WO 01/45636.Treatments schedules are within the purview of those skilled in themedicinal arts. Non-limiting examples include once daily, weekly,biweekly, monthly, or bimonthly.

EXAMPLES Example 1

Savagine and other non-selective CRFR agonists are generally noteffective in treating CRF₂R modulated disorders because these agonistsalso activate CRF₁R thereby resulting in undesirable side effects.

Table 2 reflects comparative CRF binding for native sequence fragmentsof human urocortin I (hUcnI), human urocortin II (hUroII), humanurocortin III (hUroIII), human corticotropin releasing factor (hCRF),ovine corticotropin (oCRF), and savagine (Svg) designated as SEQ ID NO:2, 4, 6, 8, 10 and 11, respectively.

TABLE 2 SEQ ID CRF₂R EC₅₀ (nM) CRF₁R EC₅₀ (nM) NO PEPTIDE (Emax %) (Emax%) 2 hUcnI  3.52 (100)   9.00 (100) 4 hUroII  3.64 (98)  >100 (9) 6hUroIII  >100 (60) >1000 (10.25) 8 hCRF 49.25 (100)  19.95 (87) 10  oCRF >100 (33)  27.35 (98) 11  Svg  6.03 (95)  17.60 (100)

Example 2

Table 3 reflects comparative CRF binding of various embodiments of theinvention.

TABLE 3 CRF₂R EC₅₀ (nM) CRF₁R EC₅₀ (nM) SEQ ID NO (Emax %) (Emax %) 131.50 (10.00) 783 (64) 3 12.13 (88) 1000 (12) 5 100 (12) 100 (9) 7 100(19) 100 (4) 9 100 (34) 100 (10) 12 9.72 (91) 928 (73) 13 3.88 (79)97.65 (69) 14 6.33 (90) 109.00 (92) 15 6.56 (97) 85.30 (86) 16 7.88 (86)136.00 (98) 17 10.20 (96) 260.50 (98) 18 5.29 (97) 106.00 (100) 19 7.42(75) 232.50 (83) 20 7.81 (99) 906.00 (60) 21 8.46 (96) 908.00 (88) 228.33 (100) 1000.00 (64) 23 10.20 (100) 1000.00 (82) 24 43.15 (100) >1000(18) 25 80.95 (81.90) 677.00 (92.35) 26 91.75 (86) >1000 (16) 27 10.70(96) 325 (100) 28 100 (15) >1000 (2) 29 16.00 (100) >1000 (79) 30 18.70(85.50) 100.45 (100) 31 30.75 (100) >1000 (18) 32 20.25 (98) 606 (94)34 >100 (45) >1000 (11) 35 15.55 (88) >1000 (74) 36 14.75 (73) >1000(33) 37 >100 (51) >1000 (5) 38 71.90 (91) >1000 (11) 39 58.17 (94) 1000(63) 40 6.95 (93) 102.5 (99) 41 18.30 (100) >1000 (43) 42 >100(88) >1000 (10) 43 >100 (67) >1000 (10) 44 19.15 (87) 943.50 (64)45 >100 (44) >1000 (7) 46 >100 (100) >1000 (17) 47 >100 (66) 1000 (12)48 100 (14) 1000 (21) 49 >100 (37) >1000 (15) 50 19.04 (94) >1000 (42)51 20.65 (100) >1000 (48) 52 >100 (10) >1000 (13) 53 >100 (95) >1000(19) 54 100 (11) 1000 (10) 55 7.95 (95) 11.60 (92) 56 50.35 (87) >100(14) 57 >100 (46) >100 (12) 58 71.60 (100) >100 (16) 59 >100 (27) >100(10) 60 >100 (44) >100 (8) 61 >100 (89) >100 (12) 63 67.35 (100) 73.15(34) 64 63.30 (94) 68.90 (57) 65 67.90 (64) >100 (16) 67 10.02 (50)44.17 (96) 68 38.55 (74) >100 (33) 69 5.85 (81) 34.50 (88) 70 18.25(82) >100 (8) 71 94.80 (56) >100 (6) 72 >100 (55) >100 (4) 73 >100(11) >100 (11) 74 54.97 (100) >100 (7) 75 >100 (52) >100 (5) 76 91.45(76) >100 (7) 77 43.35 (100) >100 (5) 78 24.65 (78) >100 (6) 79 22.30(100) >100 (8) 80 6.53 (88) >100 (55) 81 4.30 (73) 60.90 (81) 82 10.87(90) 96.20 (85) 83 1.91 (81) 52.17 (96) 84 1.77 (100) 82.23 (99) 85 2.34(100) 11.00 (84) 86 100 (8.10) 100 (4.60) 87 100 (14.65) 100 (5.30) 88100 (12.60) 100 (11.15) 89 100 (12.70) 90 100 (12.25) 100 (4.00) 91 100(7.10) 100 (4.00) 92 100 (15.85) 100 (4.60) 93 100 (6.40) 100 (5.00) 94100 (6.15) 100 (7.30) 95 100 (8.25) 100 (5.55) 96 100 (12.50) 100(16.30) 97 100 (7.60) 100 (4.25) 98 100 (5.50) 100 (4.00) 99 100 (4.35)100 (4.35) 100 100 (9.85) 100 (6.25) 101 100 (6.95) 100 (7.35) 102 100(13.50) 100 (7.80) 103 100 (4.85) 100 (5.75) 104 100 (4.50) 100 (11.10)105 100 (9.15) 100 (5.20) 106 100 (6.10) 100 (4.80) 107 12.13 (87.90)1000 (12.40) 108 79.00 (97) >100 (3) 109 11.83 (91.67) 100 (6.70) 11010.96 (100) >100 (9) 111 10.95 (99) >100 (9) 112 12.30 (100) >100 (10)113 11.30 (98) >100 (4) 114 3.42 (100) >100 (6) 115 13.60 (98) >100 (7)116 100 (26.45) 100 (4.80) 117 9.41 (98.85) 100 (7.80) 118 14.60(100) >100 (5) 119 3.57 (95) >100 (3) 120 69.90 (100) 100 (7) 121 5.67(91) >100 (3) 122 3.31 (97) 1000 (10.7) 123 3.49 (93.75) >1000 (9.60)124 3.49 (94) >100 (6) 125 4.47 (99) >100 (9) 126 13.00 (91) >100 (7)127 7.79 (94) >100 (6) 128 2.85 (98) >100 (8) 129 3.83 (92) >100 (12)130 8.57 (92) >100 (9) 131 5.25 (91) >100 (8) 132 7.53 (88) >100 (8) 13312.22 (88) >100 (4) 134 >100 (19) >100 (7) 135 >100 (76) >100 (6) 13623.40 (68) >100 (8) 137 36.90 (100) >100 (4) 138 59.00 (46) >100 (5) 13942.60 (60) >100 (4) 141 >100 (29) >100 (7) 142 9.08 (77.00) 43.45(85.35) 143 11.05 (85.50) 232.00 (100) 144 9.16 (85.53) 567 (100) 1457.80 (69.00) 196.50 (91.30) 146 8.20 (84.50) 103 (100) 147 6.75 (94.00)101.60 (96.00) 148 9.45 (51.50) 295.00 (100) 149 26.20 (95.50) 1000(40.70) 150 34.65 (70.50) 1000 (5.70) 151 36.75 (96.00) 1000 (19.90)152 >100 (19) >100 (4) 153 9.28 (97) >100 (7) 154 10.30 (100) >100 (7)155 20.60 (94) 40.65 (16) 156 9.29 (79) >100 (6) 157 42.00 (60.65) 100(91.00) 158 6.37 (89) >100 (15) 159 90.77 (62) >100 (9) 160 9.15(87) >100 (10) 161 >100 (88) >100 (77) 162 >100 (7) >100 (11) 163 4.49(96) >100 (13) 164 2.24 (92) >100 (26) 165 >100 (75) >100 (19) 166 3.7(99) 437.5 (95) 167 13.0 (100) 1000.0 (15) 168 4.9 (75) 1000.0 (52) 16917.8 (93) 978.0 (26) 170 75.0 (83) 1000.0 (9) 171 17.6 (100) 206.0 (99)172 13.5 (100) 1000.0 (68) 173 100 (65.20) 1000 (6.05) 174 4.42 (97.2)1000.0 (29.10) 175 5.42 (92.15) 1000.0 (34.8) 176 12.60 (90.25) 1000.0(26.9) 177 5.63 (97.65) 613.0 (69.2) 178 5.17 (96.45) 1000.0 (55.70) 17910.22 (92.5) 477.0 (78.90) 180 3.14 (95.7) 125.0 (99.3) 181 5.22 (97.75)154.0 (100) 182 7.21 (91.4) 409.0 (89.7) 183 7.93 (100) 415 (57.5) 1843.72 (96.45) 486.0 (77.35) 185 8.98 (100) 358.5 (95.8) 186 25.05 (100)323 (100) 187 10.3 (100) 31.6 (72) 188 14.6 (100) 162.5 (96) 189 7.0(96) 62.2 (57) 190 39.60 (31) >100 (9) 191 8.3 (100) 63.7 (65) 192 66.8(97) 562.0 (99) 193 10.1 (97) 265.5 (95) 194 5.0 (96) 106.0 (94) 19518.8 103.0 (93) 196 27.7 (97) 447.0 (96) 197 48.1 (94) 198 29.9 (100)199 8.5 (92) 706.5 (93) 200 8.8 (100) 188.5 (99) 201 5.0 (100) 99.6 (80)202 8.7 (99) 403.5 (100) 203 5.2 (94) 76.2 (86) 204 3.6 (93) 32.1 (74)205 25.0 (93) 126.5 (88) 206 30.5 (97) 696.5 (97) 207 61.4 (96) 465.5(88) 208 5.6 (88) 64.9 (81) 209 7.4 (93) 26.4 (80) 210 10.2 (97) 43.5(90) 211 59.5 (100) 826.0 (37) 212 21.3 (100) 445.0 (100) 213 22.3 (99)1000.0 (76) 214 74.30 (60) 100 (4) 215 4.0 (100) 187.5 (100) 216 9.9(90) 49.8 (98) 217 4.7 (95) 94.7 (100) 218 4.8 (96) 98.4 (100) 219 7.8(98) 80.9 (100) 220 4.1 (98) 63.6 (81) 221 8.5 (100) 236.5 (100) 222 9.4(100) 384.5 (95) 223 3.0 (92) 48.1 (81) 224 26.9 (100) 1000.0 (27) 2254.8 (89) 219.5 (97) 226 7.6 (100) 315.0 (95) 227 33.6 (95) 918.0 (18)228 7.1 (100) 275.5 (100) 229 10.3 (100) 298.0 (100) 230 8.1 (100) 219.0(100) 231 5.9 (100) 94.0 (100) 232 45.3 (100) 1000.0 (7) 233 46.1 (95)1000 (11) 234 20.6 (100) 434.3 (96) 235 25.7 (100) 806.5 (70) 236 40.4(97) 1000.0 (15) 237 22.2 (93) 1000.0 (18) 238 16.7 (100) 753.0 (88) 23913.2 (100) 587.0 (80) 240 22.0 (100) 915.0 (80) 241 12.6 (99) 307.5 (99)242 29.0 (94) 358.5 (99) 243 16.8 (100) 440.5 (96) 244 8.8 (98) 299.5(100) 245 7.5 (93) 381.0 (100) 246 38.8 (100) 1000.0 (33) 247 18.8 (100)1000.0 (34) 248 19.6 (98) 1000.0 (32) 249 12.2 (92) 1000.0 (50) 250 19.7(100) 137.5 (99) 251 11.8 (100) 926.0 (80) 252 22.3 (100) 226.5 (100)253 41.8 (86) 1000.0 (42) 254 100.0 (36) 708.0 (6) 255 7.0 (100) 33.3(84) 256 12.6 (100) 253.5 (100) 257 100 (72.60) 744.50 (83.50) 258 100(49.30) 1000 (23.65) 259 100 (64.95) 819.50 (83.60) 260 100 (89.35)834.00 (89.70) 261 100 (95.30) 274 (100) 262 100 (92.25) 408 (100) 26336.17 (93.03) 802.50 (71.95) 264 100.00 (66.30) 704.50 (100) 265 100.00(23.35) 1000.00 (9.30) 266 100.00 (19.35) 1000.00 (4.45) 267 100.00(44.20) 1000.00 (22.80) 268 100.00 (59.05) 1000.00 (14.15) 269 100.00(77.30) 1000.00 (44.30) 270 100.00 (19.30) 1000.00 (7.85) 271 48.10(68.95) 815.00 (80.65) 272 23.30 (100.00) 1000.00 (51.10) 273 31.30(100.00) 1000.00 (59.30) 274 13.80 (100.00) 508.00 (80.90) 275 46.60(100.00) 1000.00 (38.30) 276 22.10 (100.00) 1000.00 (75.70) 277 28.20(100.00) 1000.00 (39.90) 278 19.55 (100.00) 1000.00 (48.70) 279 13.10(100.00) 1000.00 (93.00) 280 100.00 (82.30) 1000.00 (11.80) 281 100.00(78.80) 1000.00 (12.20) 282 25.80 (60.75) 1000.00 (21.75) 283 10.55(71.95) 635.00 (100.00) 284 100.00 (100.00) 1000.00 (27.70) 285 13.95(97.10) 1000.00 (11.10) 286 13.50 (92.45) 1000.00 (19.20) 287 11.31(100.00) 1000.00 (51.75) 288 14.70 (100.00) 838.00 (31.65) 289 12.16(97.30) 1000.00 (69.55) 290 100.00 (100.00) 1000.00 (6.40) 291 100.00(63.75) 1000.00 (4.75) 292 100.00 (86.10) 1000.00 (7.00) 293 40.30(87.00) 520.50 (95.30) 294 100.00 (100.00) 1000.00 (13.60) 295 55.05(67.60) 1000.00 (5.30) 296 6.66 (65) 100 (4) 297 100.00 (88.30) 1000.00(27.250 298 82.00 (98.85) 1000.00 (20.05) 299 46.40 (71.55) 1000.00(10.20) 300 17.10 (100.00) 1000.00 (7.95) 301 50.45 (88.40) 690.00(84.10) 302 36.20 (100.00) 366.50 (100.00) 303 27.25 (100.00) 581.50(100.00) 304 19.30 (92.55) 115.50 (100.00) 305 35.45 (95.20) 1000.00(59.55) 306 27.55 (100.00) 608.00 (97.65) 307 5.82 (96.55) 78.40 (92.65)308 3.30 (72.80) 63.45 (100.00) 309 5.55 (99.90) 107.50 (96.35) 310 8.70(87.55) 1000.00 (45.30) 311 11.65 (100.00) 1000.00 (29.70) 312 14.05(95.00) 869.50 (62.00) 313 11.05 (95.00) 704.00 (87.100 314 10.35(99.75) 978.50 (82.40) 315 9.35 (81.70) 454.50 (100.00) 316 10.15(94.50) 221.50 (92.35) 317 9.30 (88.35) 187.50 (100.00) 318 9.95 (95.40)134.50 (92.85) 319 8.50 (95.00) 106.00 (88.30) 320 19.05 (100.00) 718.00(68.75) 321 19.55 (86.80) 1000.00 (33.80) 322 23.05 (96.45) 1000.00(10.65) 323 19.60 (100.00) 1000.00 (36.90) 324 17.20 (100.00) 1000.00(46.60) 325 11.67 (100) 100 (5) 326 33.70 (100.00) 1000.00 (30.10) 32728.40 (100.00) 1000.00 (36.50) 328 11.70 (95.70) 1000.00 (30.70) 3295.15 (98.30) 1000.00 (98.40) 330 6.00 (93.65) 1000.00 (86.80) 331 9.85(100.00) 1000.00 (78.65) 332 9.95 (100.00) 1000.00 (61.30) 333 9.85(96.90) 1000.00 (43.80) 334 13.15 (93.55) 1000.00 (82.60) 335 28.05(90.95) 1000.00 (49.45) 336 17.80 (100.00) 1000.00 (59.90) 337 23.95(86.65) 1000.00 (36.45) 338 19.30 (77.55) 1000.00 (41.10) 339 100.00(47.90) 1000.00 (13.20) 340 7.99 (100.00) 739.50 (95.95) 341 8.83(95.50) 850.50 (82.35) 342 20.25 (92.25) 1000.00 (19.65) 343 13.60(96.55) 783.00 (62.50) 344 4.30 (94.47) 650.00 (77.65) 345 39.70(100.00) 1000.00 (18.75) 346 8.48 (97.75) 1000.00 (59.00) 347 22.35(95.65) 1000.00 (48.75) 348 5.77 (90.40) 630.00 (86.05) 349 13.75 (100)1000 (44.20) 350 11.59 (98.10) 1000 (48.00) 351 12.93 (97.37) 1000(85.70) 352 8.26 (83.65) 780 (82.60) 353 4.75 (89.90) 229.50 (92.25) 3546.48 (100) 1000 (13.40) 355 6.03 (95) 18 (100) 356 83.05 (16) 1000 (9)357 6.44 (100) 331 (100) 358 5.56 (100) 99 (100) 359 23.10 (100) 230(100) 360 6.12 (93) 157 (100) 361 6.37 (99) 149 (100) 362 4.39 (100) 386(100) 363 25.15 (100) 1000 (20) 364 13.20 (100) 1000 (27) 365 23.45 (94)1000 (21) 366 100.00 (46) 1000 (9) 367 100.00 (18) 1000 (6) 368 50.35(91) 1000 (6) 369 100.00 (18) 1000 (4) 370 51.05 (75) 1000 (33) 371 6.62(97) 1000 (22) 372 14.20 (100) 1000 (13) 373 11.54 (100 1000 (9) 37415.75 (91) 1000 (11) 375 11.50 (100) 1000 (22) 376 52.55 (100) 1000 (8)377 19.25 (83) 1000 (25) 378 14.88 (100) 1000 (36) 379 70.55 (94) 1000(8) 380 19.00 (100) 1000 (16) 381 12.73 (99) 1000 (27) 382 39.45 (100)1000 (8) 383 9.31 (96) 1000 (55) 384 7.10 (97.30) 1000 (70.30) 385 10.25(100) 1000 (46) 386 8.70 (96) 1000 (78.25) 387 17.85 (100) 1000 (50.10)

Example 3

Increased In Vivo Efficacy

The peptides of the present invention exhibit extended biologicalavailability, particularly under conditions of low dosing, as comparedto known native sequences, e.g., UroII peptide fragment (SEQ ID NO: 4).

The half-life of a peptide in a subject can be determined, for example,by high performance liquid chromatography (HPLC) of serum samplescollected from the subject at various times following administration ofthe peptide. One skilled in the art would know how to select appropriateelution buffers for HPLC based on the physicochemical properties of aparticular r peptide.

A non-limiting example of an in vivo study to determine efficacy isherein described. mice are dosed by intravenous (IV) (1000 ug/kg) andsubcutaneous (SC) (1000 ug/kg) routes with a peptide of Formula (I).Blood samples are obtained at various time points (IV=0, 2, 10, 30 minand 1, 2, 4 and 6 h; and SC=0, 0.25, 0.5, 1, 2, 4, and 6 h) post dosingin microcentrifuge tubes containing sodium heparin. The blood samplesare further processed to obtain plasma which is stored at −70° C. untilanalyzed.

Plasma standards are prepared. Spiking solution of a peptide of Formula(I) covering a concentration range from 50 ng/mL to 100 μg/mL areprepared in methanol on the analysis day by serial dilution of apreviously prepared 1 mg/mL peptide of Formula (I) methanolic stocksolution. Similarly, the internal standard (ISTD), stable isotopelabeled h-Unc-II, spiking solution is prepared by serial dilution of astored 1 mg/mL ISTD stock solution to give a final concentration of 5μg/mL on the day of analysis. Working plasma standards covering a massrange from 0.5 to 100 ng are prepared by adding 10 μL of the appropriatepeptide of Formula (I) spiking solution into tubes already containing 10uL of a 5 μg/mL ISTD solution, 100 μL of dd-water and 100 μL of blankrat plasma. The working standards are prepared for analysis as describedbelow.

Quality control (QC) samples are prepared. A QC stock solution isprepared at the 50 ng/mL level by adding 25 μL of a 1 μg/mL a peptide ofFormula (I) spiking solution into 475 uL of blank, heparinized ratplasma contained in a plastic vial. Working QC samples are prepared byadding 100 uL of the QC stock solution (50 ng/mL) into tubes alreadycontaining 10 μL of a 5 μg/mL ISTD solution and 100 μL of dd-water. Theworking QC sample was prepared for analysis as described below.

Study samples are prepared. On the day of analysis, the samples arethawed at room temperature and an aliquot of the sample was added to atube already containing 10 μL of a 5 μg/mL ISTD solution, 100 μL ofdd-water and an aliquot of blank, heparinized rat plasma. The volume ofthe sample and the blank rat plasma are such that the total volume ofplasma is equal to 100 μL.

The working standards, working QC samples and study samples are preparedfor analysis by adding 400 μL of acetonitrile to tubes containing eachof these, capping, vortexing, centrifuging and isolating thesupernatant. An aliquot (300 μL) of the supernatant is dried under N₂and reconstituted in 50 μL of methanol/water (50/50).

The prepared working standards, working QC samples and study samples areanalyzed by gradient reversed-phase high performance liquidchromatography (RP-HPLC) separation followed by sample introductionthrough electron spray ionization (ESI) with mass spectroscopy/massspectroscopy (MS/MS) detection using selected reaction monitoring (SRM)in the positive ion mode. An SRM channel is monitored for h-Unc-II andthe ISTD.

The dose solutions from the pharmacokinetic study are diluted withmethanol and analyzed by RP-HPLC with ultraviolet detection. Theconcentrations of the peptide of Formula (I) in the dose solutions arecalculated by interpolation from a linear regression curve constructedfrom known standards.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

1. A non-native peptide comprising the sequence X₁X₂PSLSIDX₉PX₁₁X₁₂LLRTLLELEKTQSQRERAEQNAX₃₅IFAX₃₉V (SEQ ID NO: 531) wherein: X₁ is selected from nil and D; X₂ is selected from nil and N; X₉ is selected from L and V; X₁₁ is selected from L, and F; X₁₂ is selected from L, F, and Y; X₃₅ is selected from R, H, and Q; X₃₉ is selected from H and R; and variants thereof having at least about 95% sequence identity to said peptide.
 2. The peptide according to claim 1, wherein X₁X₂ are selected from the group consisting of DN.
 3. The peptide according to claim 1, wherein X₁₁X₁₂ are selected from the group consisting of LL, LF, FF, and LY.
 4. The peptide according to claim 2, wherein X₁₁X₁₂ are selected from the group consisting of LL, LF, FF, and LY.
 5. The peptide according to claim 1, wherein X₉ is.
 6. The peptide according to claim 1, wherein X₃₅ is.
 7. The peptide according to claim 1, wherein X₃₉ is.
 8. The peptide according to claim 1, wherein the peptide comprises a sequence selected from the group consisting of SEQ ID NO: 324, 328, 331, 333, 334, 341, 384, 385, 386, and
 387. 9. A method of preventing or treating a CRF₂R modulated disorder comprising administering to a host in need of such treatment a safe and effective amount of a peptide of claim
 1. 10. The method according to claim 9, wherein the CRF₂R modulated disorder is skeletal muscle atrophy or a form of muscular dystrophy.
 11. A pharmaceutical composition comprising: a. a safe and effective amount of a peptide of claim 1; and b. a pharmaceutically acceptable carrier.
 12. A kit for preventing or treating a CRF₂R modulated disorder comprising: a. a peptide of claim 1 in a unit dose form; and b. usage instruction.
 13. A method of preventing or treating a CRF₂R modulated disorder comprising administering to a host in need of such treatment a safe and effective amount of a peptide of claim
 8. 14. The method according to claim 13, wherein the CRF₂R modulated disorder is skeletal muscle atrophy or muscular dystrophy.
 15. A pharmaceutical composition comprising: a. a safe and effective amount of a peptide of claim 8; and b. a pharmaceutically acceptable carrier.
 16. A kit for preventing or treating a CRF₂R modulated disorder comprising: a. a peptide of claim 8 in a unit dose form; and b. usage instruction.
 17. A variant according to claim 1, having at least about 97% sequence identity to said peptide.
 18. A non-native peptide comprising the sequence of SEQ ID NO: 341, and variants thereof having at least about 95% sequence identity to said peptide.
 19. A variant according to claim 18, having at least about 97% sequence identity to said peptide.
 20. A method of preventing or treating a CRF₂R modulated disorder comprising administering to a host in need of such treatment a safe and effective amount of a peptide of claim
 18. 21. The method according to claim 20, wherein the CRF₂R modulated disorder is skeletal muscle atrophy or muscular dystrophy.
 22. A pharmaceutical composition comprising: a. a safe and effective amount of a peptide of claim 18; and b. a pharmaceutically acceptable carrier.
 23. A non-native peptide comprising the sequence of SEQ ID NO:
 341. 24. A method of preventing or treating a CRF₂R modulated disorder comprising administering to a host in need of such treatment a safe and effective amount of a peptide of claim
 23. 25. The method according to claim 24, wherein the CRF₂R modulated disorder is skeletal muscle atrophy or muscular dystrophy.
 26. A pharmaceutical composition comprising: a. a safe and effective amount of a peptide of claim 23; and b. a pharmaceutically acceptable carrier.
 27. A non-native peptide comprising the sequence: X₁X₂PSLSIDX₉PX₁₁X₁₂LLRTLLELEKTQSQRERAEQNAX₃₅IFAX₃₉V (SEQ ID NO: 531) wherein: X₁ is selected from nil and D; X₂ is selected from nil and N; X₉ is selected from L and V; X₁₁ is selected from L, and F; X₁₂ is selected from L, F, and Y; X₃₅ is selected from R, H, and Q; X₃₉ is selected from H and R.
 28. A method of preventing or treating a CRF₂R modulated disorder comprising administering to a host in need of such treatment a safe and effective amount of a peptide of claim
 27. 29. The method according to claim 28, wherein the CRF₂R modulated disorder is skeletal muscle atrophy or muscular dystrophy.
 30. A pharmaceutical composition comprising: a. a safe and effective amount of a peptide of claim 27; and b. a pharmaceutically acceptable carrier. 